Lasofoxifene treatment of breast cancer

ABSTRACT

The disclosure provides methods for treating estrogen receptor positive (ER+) cancer in women with an effective amount of lasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrug thereof. The disclosure also includes the detection of the Estrogen Receptor 1 (ESR1) gene mutations that lead to endocrine resistance and treatment of endocrine resistant ER+ cancers.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 17/073,253, filed Oct. 16, 2020, which is a continuation of U.S.application Ser. No. 16/265,109, filed Feb. 1, 2019, which is acontinuation of U.S. application Ser. No. 15/939,218, filed Mar. 28,2018, which is a continuation of U.S. application Ser. No. 15/729,320,filed Oct. 10, 2017, which claims the benefit of U.S. ProvisionalApplication Nos. 62/502,299, filed May 5, 2017; 62/457,759, filed Feb.10, 2017; and 62/406,859, filed Oct. 11, 2016, each of which isincorporated in its entirety by reference.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via Patent Center and is hereby incorporated by reference inits entirety. Said XML copy, created on Mar. 28, 2023, is named55183US_sequencelisting, and is 11,277 bytes in size.

3. BACKGROUND OF THE INVENTION

Estrogen receptor positive (ER⁺) breast cancers are a group of breastcancers that express estrogen receptor α (ERα). Approximately 70% ofbreast cancers are ER⁺ and are, therefore, treated with endocrinetherapy. Endocrine therapy has led to significant improvement in outcomeof women with ER⁺ breast cancer by lowering the level of estrogen orblocking estrogen signaling. However, its effectiveness is limited byintrinsic and acquired endocrine resistance.

Recent studies have shown evidence for the temporal selection offunctional Estrogen Receptor 1 (ESR1) gene mutations as potentialdrivers of endocrine resistance during the progression of ER⁺ breastcancer. See Jeselsohn et al., Clinical Cancer Research 20(7): 1757-1767(2014). The mutations in ESR1, the gene encoding ERα, change theconformation of the ERα protein, increase its interaction with itsco-activators, promote an active form of the receptor in absence ofhormone, and assist tumor cells in evading hormonal treatment. SeeThomas and Gustafsson, Trends in Endocrinology and Metabolism 26(9):467-476 (2015).

There thus remains a need to develop new therapeutic strategies that areeffective to treat tumors harboring mutations in ESR1, and that cantherefore be used to treat breast cancer patients who have developedendocrine resistance or who are at risk of developing endocrineresistance.

4. SUMMARY OF THE INVENTION

We engineered ERα expression constructs to express four ESR1 mutationsin the ligand binding domain (LBD) of the ERα protein, Y537S, Y537N,Y537C, and D538G, and introduced these expression constructs into cellsin culture. These mutations are found in ER⁺ metastatic breast cancerpatients who have been treated with endocrine therapy. See Jeselsohn etal., Nature Reviews Clinical Oncology 12(10): 573-583 (2015); Jeselsohnet al., Clinical Cancer Research 20(7): 1757-1767 (2014); Robinson etal., Nature Genetics 45(12): 1446-1451(2013); Thomas and Gustafsson,Trends in Endocrinology and Metabolism 26(9): 467-476 (2015); and Toy etal., Nature Genetics 45(12): 1439-1445 (2013).

Using an estrogen receptor-responsive reporter construct, we confirmedin an ovarian cell line and in a breast cancer cell line that allmutants are constitutively active as compared to wild type ERα. We thentreated the cells with lasofoxifene, a selective ER modulator (SERM),and found that lasofoxifene effectively inhibited the transcriptionalactivity of the ERα LBD mutants in a dose-response manner, atconcentrations that are clinically achievable.

In a second series of experiments, we confirmed that lasofoxifene isable to reduce viability of the breast cancer cell line MCF7 stablytransfected with either the Y537S or D538G ESR1 mutant receptor, atclinically achievable concentrations.

Accordingly, in a first aspect, a method of treating locally advanced ormetastatic breast cancer in women is presented. The method comprisesselecting for treatment a patient who has been diagnosed with estrogenreceptor positive (ER⁺) locally advanced or metastatic breast cancer,and administering to the selected patient an effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof.

In various embodiments, the selected patient has previously been treatedwith one or more lines of endocrine therapy. In certain embodiments, thepatient has previously been treated with a plurality of lines ofendocrine therapy.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is a selective ER modulator (SERM). Incertain embodiments, the SERM is tamoxifen, raloxifene, bazedoxifene,toremifene, or ospemifene.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is a selective ER degrader (SERD). Incertain embodiments, the SERD is fulvestrant, RAD1901, ARN-810(GDC-0810), or AZD9496.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is an aromatase inhibitor. In certainembodiments, the aromatase inhibitor is exemestane (Aromasin®),letrozole (Femara®), or anastrozole (Arimidex®).

In some embodiments, the patient has disease progression after endocrinetherapy. In some embodiments, the patient is resistant to endocrinetherapy.

In various embodiments, the patient's cancer has at least one gain offunction missense mutation within the ligand binding domain (LBD) of theEstrogen Receptor 1 (ESR1) gene. In some embodiments, the patient haspreviously been determined to have at least one gain of functionmissense mutation within the ligand binding domain (LBD) of the EstrogenReceptor 1 (ESR1) gene. In certain embodiments, the method furthercomprises the earlier step of: determining that the patient has at leastone gain of function missense mutation within the ligand binding domain(LBD) of the Estrogen Receptor 1 (ESR1) gene.

In some embodiments, the at least one of gain of function missensemutation is in any one of amino acids D538, Y537, L536, P535, V534,S463, V392, or E380.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid D538. In some preferred embodiments themutation is D538G.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid Y537. In some embodiments, the mutation isY537S, Y537N, Y537C, or Y537Q. In some preferred embodiments, themutation is Y537C.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid L536. In some embodiments, the mutation isL536R or L536Q.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid P535. In some embodiments, the mutation isP535H.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid V534. In some embodiments, the mutation isV534E.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid S463. In some embodiments, the mutation isS463P.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid V392. In some embodiments, the mutation isV392I.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid E380. In some embodiments, the mutation isE380Q.

In some embodiments, the serum estradiol level of the patient is atleast 0.35 ng/dL. In some embodiments, the serum estradiol level of thepatient is about 0.30 ng/dL to about 0.35 ng/dL. In some embodiments,the serum estradiol level of the patient is about 0.25 ng/dL to about0.30 ng/dL.

In various embodiments, lasofoxifene is administered to the selected ER⁺locally advanced or metastatic breast cancer patient as lasofoxifenetartrate. In various embodiments, lasofoxifene is administered by oral,intravenous, transdermal, vaginal topical, or vaginal ringadministration. In certain embodiments, lasofoxifene is administered byoral administration. In some of these embodiments, lasofoxifene isadministered at about 0.5 mg/day per os (p.o.) to about 10 mg/day peros. In certain embodiments, lasofoxifene is administered at about 0.5mg/day per os to about 5 mg/day per os. In certain embodiments,lasofoxifene is administered at about 1 mg/day per os to about 5 mg/dayper os. In certain embodiments, lasofoxifene is administered at about 1mg/day per os. In certain embodiments, lasofoxifene is administered atabout 5 mg/day per os. In various embodiments, lasofoxifene isadministered once every day, once every two days, once every three days,once every four days, once every five days, once every six days, onceevery week, once every two weeks, once every three weeks, or once everymonth.

In certain embodiments, the method further comprises treating thepatient with at least one additional endocrine therapy. In someembodiments, the patient is treated with the additional endocrinetherapy at original doses. In some other embodiments, the patient istreated with the additional endocrine therapy at doses higher thanoriginal doses. In certain embodiments, the additional endocrine therapyis treatment with a selective ER modulator (SERM) other thanlasofoxifene. In certain embodiments, the additional endocrine therapyis treatment with a selective ER degrader (SERD). In certainembodiments, the additional endocrine therapy is treatment with anaromatase inhibitor.

In various embodiments, the method further comprises administering tothe ER⁺ locally advanced or metastatic breast cancer patient aneffective amount of cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Incertain embodiments, CDK4/6 inhibitor is palbociclib, abemaciclib, orribociclib. In some embodiments, the method further comprisesadministering to the patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor. In certain embodiments, the mTOR inhibitoris Everolimus. In some embodiments, the method further comprisesadministering to the patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor. Insome embodiments, the method further comprises administering to thepatient an effective amount of human epidermal growth factor receptor 2(HER2) inhibitor. In certain embodiments, the HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®). Insome embodiments, the method further comprises administering to thepatient an effective amount of a histone deacetylase (HDAC) inhibitor.In some of these embodiments, the HDAC inhibitor is vorinostat(Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®), panobinostat(Farydak®), belinostat (Beleodaq®, PXD101), valproic acid (Depakote®,Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat (PCI-24781),entinostat (MS-275), pracinostat (SB939), resminostat (4SC-201),givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin, CUDC-101,AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745, rocilinostat(ACY-1215), or sulforaphane. In some embodiments, the method furthercomprises administering to the patient an effective amount of acheckpoint inhibitor. In some of these embodiments, the checkpointinhibitor is an antibody specific for programmed cell death protein 1(PD-1), programmed death-ligand 1 (PD-L1), or cytotoxicT-lymphocyte-associated protein 4 (CTLA-4). In certain embodiments, thePD-1 antibody is pembrolizumab (Keytruda®) or nivolumab (Opdivo®). Incertain embodiments, the CTLA-4 antibody is ipilimumab (Yervoy®). Insome embodiments, the method further comprises administering to thepatient an effective amount of cancer vaccine.

In some embodiments, the patient is premenopausal. In certainembodiments, the patient has locally advanced or metastatic ER+/HER2−breast cancer. In some of these embodiments, the patient has progressedon her first hormonal treatment while on a non-steroid aromataseinhibitor (AI), fulvestrant, AI in combination with a CDK4/6 inhibitor,or fulvestrant in combination with a CDK4/6 inhibitor.

In some embodiments, the patient is perimenopausal. In certainembodiments, the patient has locally advanced or metastatic ER+/HER2−breast cancer. In some of these embodiments, the patient has progressedon her first hormonal treatment while on a non-steroid aromataseinhibitor (AI), fulvestrant, AI in combination with a CDK4/6 inhibitor,or fulvestrant in combination with a CDK4/6 inhibitor.

In some embodiments, the patient is postmenopausal. In certainembodiments, the patient has locally advanced or metastatic ER+/HER2−breast cancer. In some of these embodiments, the patient has progressedon her first hormonal treatment while on a non-steroid aromataseinhibitor (AI), fulvestrant, AI in combination with a CDK4/6 inhibitor,or fulvestrant in combination with a CDK4/6 inhibitor.

In another aspect, a method of treating primary breast cancer in womenis presented. The method comprises selecting for treatment a patient whohas been diagnosed with estrogen receptor positive (ER⁺) primary breastcancer, and administering to the selected patient an effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof.

In various embodiments, lasofoxifene is administered to the selected ER⁺primary breast cancer patient as lasofoxifene tartrate. In someembodiments, lasofoxifene is administered by oral, intravenous,transdermal, vaginal topical, or vaginal ring administration. In certainembodiments, lasofoxifene is administered by oral administration. Insome of these embodiments, lasofoxifene is administered at about 0.5mg/day per os to about 10 mg/day per os. In certain embodiments,lasofoxifene is administered at about 0.5 mg/day per os to about 5mg/day per os. In certain embodiments, lasofoxifene is administered atabout 1 mg/day per os to about 5 mg/day per os. In certain embodiments,lasofoxifene is administered at about 1 mg/day per os. In certainembodiments, lasofoxifene is administered at about 5 mg/day per os. Invarious embodiments, lasofoxifene is administered once every day, onceevery two days, once every three days, once every four days, once everyfive days, once every six days, once every week, once every two weeks,once every three weeks, or once every month.

In various embodiments, the method of treating ER⁺ primary breast cancerfurther comprises treating the patient with at least one additionalendocrine therapy. In some embodiments, the patient is treated with theadditional endocrine therapy at original doses. In some otherembodiments, the patient is treated with the additional endocrinetherapy at doses higher than original doses. In certain embodiments, theadditional endocrine therapy is treatment with a selective ER modulator(SERM) other than lasofoxifene. In certain embodiments, the additionalendocrine therapy is treatment with a selective ER degrader (SERD). Incertain embodiments the additional endocrine therapy is treatment withan aromatase inhibitor.

In various embodiments, the method further comprises administering tothe ER⁺ primary breast cancer patient an effective amount ofcyclin-dependent kinase 4/6 (CDK4/6) inhibitor. In certain embodiments,CDK4/6 inhibitor is palbociclib, abemaciclib, or ribociclib. In someembodiments, the method further comprises administering to the patientan effective amount of mammalian target of rapamycin (mTOR) inhibitor.In certain embodiments, the mTOR inhibitor is Everolimus. In someembodiments, the method further comprises administering to the patientan effective amount of phosphoinositide 3-kinase (PI3K) inhibitor orheat shock protein 90 (HSP90) inhibitor. In some embodiments, the methodfurther comprises administering to the patient an effective amount ofhuman epidermal growth factor receptor 2 (HER2) inhibitor. In certainembodiments, the HER2 inhibitor is trastuzumab (Herceptin®) orado-trastuzumab emtansine (Kadcyla®). In some embodiments, the methodfurther comprises administering to the patient an effective amount of ahistone deacetylase (HDAC) inhibitor. In some of these embodiments, theHDAC inhibitor is vorinostat (Zolinza®), romidepsin (Istodax®),chidamide (Epidaza®), panobinostat (Farydak®), belinostat (Beleodaq®,PXD101), valproic acid (Depakote®, Depakene®, Stavzor®), mocetinostat(MGCD0103), abexinostat (PCI-24781), entinostat (MS-275), pracinostat(SB939), resminostat (4SC-201), givinostat (ITF2357), quisinostat(JNJ-26481585), kevetrin, CUDC-101, AR-42, tefinostat (CHR-2835),CHR-3996, 4SC202, CG200745, rocilinostat (ACY-1215), or sulforaphane. Insome embodiments, the method further comprises administering to thepatient an effective amount of a checkpoint inhibitor. In some of theseembodiments, the checkpoint inhibitor is an antibody specific forprogrammed cell death protein 1 (PD-1), programmed death-ligand 1(PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Incertain embodiments, the PD-1 antibody is pembrolizumab (Keytruda®) ornivolumab (Opdivo®). In certain embodiments, the CTLA-4 antibody isipilimumab (Yervoy®). In some embodiments, the method further comprisesadministering to the patient an effective amount of cancer vaccine.

In certain embodiments, the patient is premenopausal. In certainembodiments, the patient is perimenopausal. In certain embodiments, thepatient is postmenopausal.

In another aspect, a method of adjuvant therapy for estrogen receptorpositive (ER+) breast cancer is presented. The method comprisesadministering to a patient who has received primary treatment for ER+breast cancer an effective amount of lasofoxifene, a pharmaceuticallyacceptable salt thereof, or a prodrug thereof, in combination with anaromatase inhibitor.

In some embodiments, lasofoxifene is administered continuously duringthe administration of the aromatase inhibitor. In some embodiments,lasofoxifene is administered cyclically during the administration of thearomatase inhibitor. In certain embodiments, the dosing regimen oflasofoxifene is different from the dosing regimen of the aromataseinhibitor.

In various embodiments, lasofoxifene is administered as lasofoxifenetartrate as adjuvant therapy in combination with an aromatase inhibitor.In some embodiments, the aromatase inhibitor is exemestane (Aromasin®),letrozole (Femara®), or anastrozole (Arimidex®). In some embodiments,lasofoxifene is administered by oral, intravenous, transdermal, vaginaltopical, or vaginal ring administration. In certain embodiments,lasofoxifene is administered by oral administration. In some of theseembodiments, lasofoxifene is administered at about 0.5 mg/day per os toabout 10 mg/day per os. In certain embodiments, lasofoxifene isadministered at about 0.5 mg/day per os to about 5 mg/day per os. Incertain embodiments, lasofoxifene is administered at about 1 mg/day peros to about 5 mg/day per os. In certain embodiments, lasofoxifene isadministered at about 1 mg/day per os. In certain embodiments,lasofoxifene is administered at about 5 mg/day per os. In variousembodiments, lasofoxifene is administered once every day, once every twodays, once every three days, once every four days, once every five days,once every six days, once every week, once every two weeks, once everythree weeks, or once every month.

In various embodiments, the method of adjuvant therapy for estrogenreceptor positive (ER+) breast cancer further comprises treating thepatient with at least one additional endocrine therapy. In certainembodiments, the additional endocrine therapy is treatment with aselective ER degrader (SERD).

In various embodiments, the method of adjuvant therapy for estrogenreceptor positive (ER+) breast cancer further comprises administering tothe patient an effective amount of cyclin-dependent kinase 4/6 (CDK4/6)inhibitor. In certain embodiments, CDK4/6 inhibitor is palbociclib,abemaciclib, or ribociclib. In some embodiments, the method furthercomprises administering to the patient an effective amount of mammaliantarget of rapamycin (mTOR) inhibitor. In certain embodiments, the mTORinhibitor is Everolimus. In some embodiments, the method furthercomprises administering to the patient an effective amount ofphosphoinositide 3-kinase (PI3K) inhibitor or heat shock protein 90(HSP90) inhibitor. In some embodiments, the method further comprisesadministering to the patient an effective amount of human epidermalgrowth factor receptor 2 (HER2) inhibitor. In certain embodiments, theHER2 inhibitor is trastuzumab (Herceptin®) or ado-trastuzumab emtansine(Kadcyla®). In some embodiments, the method further comprisesadministering to the patient an effective amount of a histonedeacetylase (HDAC) inhibitor. In some of these embodiments, the HDACinhibitor is vorinostat (Zolinza®), romidepsin (Istodax®), chidamide(Epidaza®), panobinostat (Farydak®), belinostat (Beleodaq®, PXD101),valproic acid (Depakote®, Depakene®, Stavzor®), mocetinostat (MGCD0103),abexinostat (PCI-24781), entinostat (MS-275), pracinostat (SB939),resminostat (4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585),kevetrin, CUDC-101, AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202,CG200745, rocilinostat (ACY-1215), or sulforaphane. In some embodiments,the method further comprises administering to the patient an effectiveamount of a checkpoint inhibitor. In some of these embodiments, thecheckpoint inhibitor is an antibody specific for programmed cell deathprotein 1 (PD-1), programmed death-ligand 1 (PD-L1), or cytotoxicT-lymphocyte-associated protein 4 (CTLA-4). In certain embodiments, thePD-1 antibody is pembrolizumab (Keytruda®) or nivolumab (Opdivo®). Incertain embodiments, the CTLA-4 antibody is ipilimumab (Yervoy®). Insome embodiments, the method further comprises administering to thepatient an effective amount of cancer vaccine.

In some embodiments, lasofoxifene is administered in an amount and on aschedule sufficient to improve bone mass. In some embodiments,lasofoxifene is administered in an amount and on a schedule sufficientto improve symptoms of VVA.

In certain embodiments, the patient is premenopausal. In certainembodiments, the patient is perimenopausal. In certain embodiments, thepatient is postmenopausal.

In another aspect, a method of treating cancers other than breast cancerin women is presented. The method comprises selecting for treatment apatient who has been diagnosed with estrogen receptor positive (ER⁺)cancer, other than breast cancer, and has at least one gain of functionmutations in the Estrogen Receptor 1 (ESR1) gene, and administering tothe selected patient an effective amount of lasofoxifene, apharmaceutically acceptable salt thereof, or a prodrug thereof. In someembodiments, the patient has been diagnosed with ER⁺ ovarian cancer. Insome other embodiments, the patient has been diagnosed with ER⁺ lungcancer.

In various embodiments, lasofoxifene is administered to the selectedpatient with ER⁺ cancer, other than breast cancer, as lasofoxifenetartrate. In some embodiments, lasofoxifene is administered by oral,intravenous, transdermal, vaginal topical, or vaginal ringadministration. In certain embodiments, lasofoxifene is administered byoral administration. In some of these embodiments, lasofoxifene isadministered at about 0.5 mg/day per os to about 10 mg/day per os. Incertain embodiments, lasofoxifene is administered at about 0.5 mg/dayper os to about 5 mg/day per os. In certain embodiments, lasofoxifene isadministered at about 1 mg/day per os to about 5 mg/day per os. Incertain embodiments, lasofoxifene is administered at about 1 mg/day peros. In certain embodiments, lasofoxifene is administered at about 5mg/day per os. In various embodiments, lasofoxifene is administered onceevery day, once every two days, once every three days, once every fourdays, once every five days, once every six days, once every week, onceevery two weeks, once every three weeks, or once every month.

In various embodiments, the method of treating ER⁺ cancer, other thanbreast cancer, further comprises treating the patient with at least oneadditional endocrine therapy. In some embodiments, the patient istreated with the additional endocrine therapy at original doses. In someother embodiments, the patient is treated with the additional endocrinetherapy at doses higher than original doses. In certain embodiments, theadditional endocrine therapy is treatment with a selective ER modulator(SERM) other than lasofoxifene. In certain embodiments, the additionalendocrine therapy is treatment with a selective ER degrader (SERD). Incertain embodiments the additional endocrine therapy is treatment withan aromatase inhibitor.

In various embodiments, the method further comprises administering tothe patient with ER⁺ cancer, other than breast cancer, an effectiveamount of cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. In certainembodiments, CDK4/6 inhibitor is palbociclib, abemaciclib, orribociclib. In some embodiments, the method further comprisesadministering to the patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor. In certain embodiments, the mTOR inhibitoris Everolimus. In some embodiments, the method further comprisesadministering to the patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor. Insome embodiments, the method further comprises administering to thepatient an effective amount of human epidermal growth factor receptor 2(HER2) inhibitor. In certain embodiments, the HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®). Insome embodiments, the method further comprises administering to thepatient an effective amount of a histone deacetylase (HDAC) inhibitor.In some of these embodiments, the HDAC inhibitor is vorinostat(Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®), panobinostat(Farydak®), belinostat (Beleodaq®, PXD101), valproic acid (Depakote®,Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat (PCI-24781),entinostat (MS-275), pracinostat (SB939), resminostat (4SC-201),givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin, CUDC-101,AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745, rocilinostat(ACY-1215), or sulforaphane. In some embodiments, the method furthercomprises administering to the patient an effective amount of acheckpoint inhibitor. In some of these embodiments, the checkpointinhibitor is an antibody specific for programmed cell death protein 1(PD-1), programmed death-ligand 1 (PD-L1), or cytotoxicT-lymphocyte-associated protein 4 (CTLA-4). In certain embodiments, thePD-1 antibody is pembrolizumab (Keytruda®) or nivolumab (Opdivo®). Incertain embodiments, the CTLA-4 antibody is ipilimumab (Yervoy®). Insome embodiments, the method further comprises administering to thepatient an effective amount of cancer vaccine.

In certain embodiments, the patient is premenopausal. In certainembodiments, the patient is perimenopausal. In certain embodiments, thepatient is postmenopausal.

In another aspect, a method of treating a female patient suffering frombreast cancer who is at risk of acquiring a gain of function missensemutation within the ligand binding domain (LBD) of the Estrogen Receptor1 (ESR1) gene is presented. The method comprises administering to thefemale patient an effective amount of lasofoxifene, a pharmaceuticallyacceptable salt thereof, or a prodrug thereof.

In another aspect, a method of treating a female patient suffering frombreast cancer who is at risk of acquiring resistance to endocrinetherapy is presented. The endocrine therapy is optionally (i) selectiveER modulator (SERM) therapy, (ii) selective ER degrader (SERD) therapy,(iii) aromatase inhibitor (AI) therapy, or (iv) any combination of (i),(ii) and/or (iii). The method comprises administering to the femalepatient an effective amount of lasofoxifene, a pharmaceuticallyacceptable salt thereof, or a prodrug thereof.

In some embodiments, the patient has primary breast cancer. In some ofthese embodiments, the primary breast cancer is locally advanced.

In various embodiments, the patient has been treated with endocrinetherapy, optionally wherein the endocrine therapy is (i) selective ERmodulator (SERM) therapy, (ii) selective ER degrader (SERD) therapy,(iii) aromatase inhibitor (AI) therapy, or (iv) any combination of (i),(ii) and/or (iii).

In another aspect, a method of treating a female patient suffering fromestrogen receptor positive (ER+) primary breast cancer is presented. Themethod comprises administering to a female patient an effective amountof lasofoxifene, a pharmaceutically acceptable salt thereof, or aprodrug thereof.

In some embodiments, the patient is at risk of acquiring resistance toendocrine therapy, optionally wherein the endocrine therapy is (i)selective ER modulator (SERM) therapy, (ii) selective ER degrader (SERD)therapy, (iii) aromatase inhibitor (AI) therapy, or (iv) any combinationof (i), (ii) and/or (iii).

In certain embodiments, the primary breast cancer is locally advanced.

In some embodiments, the patient has been treated with endocrinetherapy, optionally wherein the endocrine therapy is (i) selective ERmodulator (SERM) therapy, (ii) selective ER degrader (SERD) therapy,(iii) aromatase inhibitor (AI) therapy, or (iv) any combination of (i),(ii) and/or (iii).

In another aspect, a method of treating a female patient suffering fromestrogen receptor positive (ER+) locally advanced or metastatic breastcancer is presented. The method comprises administering to a femalepatient an effective amount of lasofoxifene, a pharmaceuticallyacceptable salt thereof, or a prodrug thereof.

In various embodiments, the selected patient has previously been treatedwith one or more lines of endocrine therapy. In certain embodiments, thepatient has previously been treated with a plurality of lines ofendocrine therapy.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is a selective ER modulator (SERM). Incertain embodiments, the SERM is tamoxifen, raloxifene, bazedoxifene,toremifene, or ospemifene.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is a selective ER degrader (SERD). Incertain embodiments, the SERD is fulvestrant, RAD1901, ARN-810(GDC-0810), or AZD9496.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is an aromatase inhibitor. In certainembodiments, the aromatase inhibitor is exemestane (Aromasin®),letrozole (Femara®), or anastrozole (Arimidex®).

In some embodiments, the patient has disease progression after endocrinetherapy. In some embodiments, the patient is resistant to endocrinetherapy.

In various embodiments, the patient's cancer has at least one gain offunction missense mutation within the ligand binding domain (LBD) of theEstrogen Receptor 1 (ESR1) gene. In some embodiments, the patient haspreviously been determined to have at least one gain of functionmissense mutation within the ligand binding domain (LBD) of the EstrogenReceptor 1 (ESR1) gene. In certain embodiments, the method furthercomprises the earlier step of: determining that the patient has at leastone gain of function missense mutation within the ligand binding domain(LBD) of the Estrogen Receptor 1 (ESR1) gene.

In some embodiments, the at least one of gain of function missensemutation is in any one of amino acids D538, Y537, L536, P535, V534,S463, V392, or E380.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid D538. In some preferred embodiments themutation is D538G.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid Y537. In some embodiments, the mutation isY537S, Y537N, Y537C, or Y537Q. In some preferred embodiments, themutation is Y537C.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid L536. In some embodiments, the mutation isL536R or L536Q.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid P535. In some embodiments, the mutation isP535H.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid V534. In some embodiments, the mutation isV534E.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid S463. In some embodiments, the mutation isS463P.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid V392. In some embodiments, the mutation isV392I.

In certain embodiments, the at least one gain of function missensemutation is in the amino acid E380. In some embodiments, the mutation isE380Q.

In various embodiments, lasofoxifene is administered to the selected ER⁺locally advanced or metastatic breast cancer patient as lasofoxifenetartrate. In various embodiments, lasofoxifene is administered by oral,intravenous, transdermal, vaginal topical, or vaginal ringadministration. In certain embodiments, lasofoxifene is administered byoral administration. In some of these embodiments, lasofoxifene isadministered at about 0.5 mg/day per os (p.o.) to about 10 mg/day peros. In certain embodiments, lasofoxifene is administered at about 0.5mg/day per os to about 5 mg/day per os. In certain embodiments,lasofoxifene is administered at about 1 mg/day per os to about 5 mg/dayper os. In certain embodiments, lasofoxifene is administered at about 1mg/day per os. In certain embodiments, lasofoxifene is administered atabout 5 mg/day per os. In various embodiments, lasofoxifene isadministered once every day, once every two days, once every three days,once every four days, once every five days, once every six days, onceevery week, once every two weeks, once every three weeks, or once everymonth.

In certain embodiments, the method further comprises treating thepatient with at least one additional endocrine therapy. In someembodiments, the patient is treated with the additional endocrinetherapy at original doses. In some other embodiments, the patient istreated with the additional endocrine therapy at doses higher thanoriginal doses. In certain embodiments, the additional endocrine therapyis treatment with a selective ER modulator (SERM) other thanlasofoxifene. In certain embodiments, the additional endocrine therapyis treatment with a selective ER degrader (SERD). In certainembodiments, the additional endocrine therapy is treatment with anaromatase inhibitor.

In various embodiments, the method further comprises administering tothe ER⁺ locally advanced or metastatic breast cancer patient aneffective amount of cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Incertain embodiments, CDK4/6 inhibitor is palbociclib, abemaciclib, orribociclib. In some embodiments, the method further comprisesadministering to the patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor. In certain embodiments, the mTOR inhibitoris Everolimus. In some embodiments, the method further comprisesadministering to the patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor. Insome embodiments, the method further comprises administering to thepatient an effective amount of human epidermal growth factor receptor 2(HER2) inhibitor. In certain embodiments, the HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®). Insome embodiments, the method further comprises administering to thepatient an effective amount of a histone deacetylase (HDAC) inhibitor.In some of these embodiments, the HDAC inhibitor is vorinostat(Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®), panobinostat(Farydak®), belinostat (Beleodaq®, PXD101), valproic acid (Depakote®,Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat (PCI-24781),entinostat (MS-275), pracinostat (SB939), resminostat (4SC-201),givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin, CUDC-101,AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745, rocilinostat(ACY-1215), or sulforaphane. In some embodiments, the method furthercomprises administering to the patient an effective amount of acheckpoint inhibitor. In some of these embodiments, the checkpointinhibitor is an antibody specific for programmed cell death protein 1(PD-1), programmed death-ligand 1 (PD-L1), or cytotoxicT-lymphocyte-associated protein 4 (CTLA-4). In certain embodiments, thePD-1 antibody is pembrolizumab (Keytruda®) or nivolumab (Opdivo®). Incertain embodiments, the CTLA-4 antibody is ipilimumab (Yervoy®). Insome embodiments, the method further comprises administering to thepatient an effective amount of cancer vaccine.

In some embodiments, the patient is premenopausal. In certainembodiments, the patient has locally advanced or metastatic ER+/HER2−breast cancer. In some of these embodiments, the patient has progressedon her first hormonal treatment while on a non-steroid aromataseinhibitor (AI), fulvestrant, AI in combination with a CDK4/6 inhibitor,or fulvestrant in combination with a CDK4/6 inhibitor.

In some embodiments, the patient is perimenopausal. In certainembodiments, the patient has locally advanced or metastatic ER+/HER2−breast cancer. In some of these embodiments, the patient has progressedon her first hormonal treatment while on a non-steroid aromataseinhibitor (AI), fulvestrant, AI in combination with a CDK4/6 inhibitor,or fulvestrant in combination with a CDK4/6 inhibitor.

In some embodiments, the patient is postmenopausal. In certainembodiments, the patient has locally advanced or metastatic ER+/HER2−breast cancer. In some of these embodiments, the patient has progressedon her first hormonal treatment while on a non-steroid aromataseinhibitor (AI), fulvestrant, AI in combination with a CDK4/6 inhibitor,or fulvestrant in combination with a CDK4/6 inhibitor.

5. BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1A and FIG. 1B show the effects of lasofoxifene on ESR1 ligandbinding domain (“LBD”) mutations in Caov2 ovarian carcinoma cells, withFIG. 1A demonstrating that the mutant receptors are constitutivelyactive and do not respond to 17-β estradiol (“E2”), and FIG. 1Bdemonstrating that lasofoxifene inhibits the mutant receptor activity ina dose-response manner.

FIG. 2A and FIG. 2B show the effects of lasofoxifene on ESR1 LBDmutations in SKBR3 breast adenocarcinoma cells, with FIG. 2Ademonstrating that the mutant receptors are constitutively active and donot respond to 17-β estradiol (E2), and FIG. 2B demonstrating thatlasofoxifene inhibits the mutant receptor activity in a dose-responsemanner.

FIG. 3A and FIG. 3B show the effects of lasofoxifene on ESR1 LBDmutations in stably transfected MCF7 breast cancer cells, with FIG. 3Ademonstrating that lasofoxifene inhibits the Y537S mutant receptoractivity with increasing dose titration, and FIG. 3B demonstrating thatlasofoxifene inhibits the D538G mutant receptor activity with increasingdose titration.

6. DETAILED DESCRIPTION OF THE INVENTION

Endocrine therapy is often used for treatment and prevention of ER⁺breast cancers. Different types of endocrine therapy include selectiveER modulators (SERMs), such as tamoxifen; selective ER degraders(SERDs), such as fulvestrant; and aromatase inhibitors (AIs). Althoughendocrine therapy has led to a significant improvement in outcome forwomen with ER⁺ breast cancer, its effectiveness is limited by intrinsicand acquired endocrine resistance. Recent studies on the mechanism ofendocrine resistance have demonstrated that in some cases EstrogenReceptor 1 (ESR1) gene mutations lead to the conformational change ofthe ERα protein towards a constitutively active state and result inligand-independent activity that is relatively resistant to tamoxifen,fulvestrant, and estrogen deprivation. See Jeselsohn et al., ClinicalCancer Research 20(7): 1757-1767 (2014).

Lasofoxifene is a nonsteroidal selective ER modulator (SERM). It hashigh binding affinity for the estrogen receptor and acts as atissue-selective estrogen agonist or antagonist. In the double-blind,placebo-controlled, randomized Postmenopausal Evaluation andRisk-Reduction with Lasofoxifene (PEARL) trial, lasofoxifene was foundto reduce the risk of osteoporosis. See Cummings et al., The New EnglandJournal of Medicine 326(8): 686-696 (2010). In the PEARL trial, it wasalso found that lasofoxifene reduced the risk of breast cancer inpost-menopausal women with osteoporosis. See LaCroix et al., Journal ofthe National Cancer Institute 102(22): 1706-1715 (2010). However, theeffect of lasofoxifene as a treatment for breast cancer, and its effecton cancers with endocrine resistance, has not previously beendetermined.

Using cell lines with engineered mutations in the ESR1 gene, wediscovered that lasofoxifene inhibits the mutant receptor activity in adose-responsive manner at concentrations that can be achievedclinically, newly making possible methods of treating ER⁺ locallyadvanced or metastatic breast cancer, ER⁺ primary breast cancer, andother ER⁺ cancers, including cancers having ESR1 mutations, usinglasofoxifene, whose effectiveness is not precluded by endocrineresistance.

6.1. METHODS OF TREATMENT

Accordingly, in a first aspect, disclosed herein are methods of treatingcancers in women, comprising selecting for treatment a patient who hasbeen diagnosed with estrogen receptor positive (ER⁺) cancer. Theselected patient is treated with an effective amount of lasofoxifene, apharmaceutically acceptable salt thereof, or a prodrug thereof.

6.1.1. Patient with ER⁺ Cancer

In various embodiments, the patient has been diagnosed with ER⁺ cancerby immunohistochemistry (IHC) performed on a sample of the patient'scancer. In some embodiments, the patient has been diagnosed with locallyadvanced or metastatic ER⁺ breast cancer. In some embodiments, thepatient has been diagnosed with ER⁺ primary breast cancer. In someembodiments, the patient has been diagnosed with an ER⁺ cancer otherthan breast cancer. In some of these embodiments, the patient has beendiagnosed with ER⁺ ovarian cancer. In some of these embodiments, thepatient has been diagnosed with ER⁺ lung cancer.

In some embodiments, cells of the patient's cancer have acquired a gainof function missense mutation within the ligand binding domain (LBD) ofthe Estrogen Receptor 1 (ESR1) gene.

In some embodiments, the patient is at risk of acquiring resistance toendocrine therapy. In particular embodiments, the patient is at risk ofacquiring resistance to endocrine therapy due to the increasedexpression of estrogen receptor. In particular embodiments, the patientis at risk of acquiring resistance to endocrine therapy due to theincreased expression of co-activators of estrogen receptor. Inparticular embodiments, the patient is at risk of acquiring resistanceto endocrine therapy due to increased phosphorylation level and activityof estrogen receptor and its co-activators. In particular embodiments,the patient is at risk of acquiring resistance to endocrine therapy dueto change of tumor microenvironment and other host related factors. Insome preferred embodiments, the patient is at risk of acquiringresistance to endocrine therapy due to mutations in the EstrogenReceptor 1 (ESR1) gene.

In some of these embodiments, the endocrine therapy to which the patientis at risk of acquiring resistance is (i) selective ER modulator (SERM)therapy, (ii) selective ER degrader (SERD) therapy, (iii) aromataseinhibitor therapy (AI), or (iv) any combination of (i), (ii) and/or(iii).

6.1.2. Previous Treatment with Endocrine Therapy

In various embodiments, the ER⁺ cancer patient has previously beentreated with one or more lines of endocrine therapy. In certainembodiments, the patient has previously been treated with one line ofendocrine therapy. In certain other embodiments, the patient haspreviously been treated with a plurality of lines of endocrine therapy.In some embodiments, the patient has previously been treated with twolines of endocrine therapy. In some embodiments, the patient haspreviously been treated with three lines of endocrine therapy. In someembodiments, the patient has previously been treated with four or morelines of endocrine therapy.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is a selective ER modulator (SERM). In someembodiments, the selective ER modulator is selected from tamoxifen,raloxifene, bazedoxifene, toremifene, and ospemifene. In certainembodiments, the selective ER modulator is tamoxifen.

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is a selective ER degrader (SERD). Invarious embodiments, the selective ER degrader binds to the estrogenreceptor and leads to the proteasomal degradation of the receptor. Insome embodiments, the selective ER degrader is selected fromfulvestrant, RAD1901, ARN-810 (GDC-0810), and AZD9496. In certainembodiments, the selective ER degrader is fulvestrant.

In some embodiments, the endocrine therapy with which the patient haspreviously been treated is an aromatase inhibitor (AI). In variousembodiments, the aromatase inhibitor blocks the production of estrogen.In some embodiments, the aromatase inhibitor is selected from exemestane(Aromasin®), letrozole (Femara®), and anastrozole (Arimidex®).

In some embodiments, the endocrine therapy that the patient haspreviously been treated with is ovarian suppression. In certainembodiments, ovarian suppression is achieved by oophorectomy. In certainembodiments, ovarian suppression is achieved by administration of a GnRHantagonist.

In certain embodiments, the patient's cancer has relapsed or progressedafter the previous endocrine therapy treatment. In some embodiments, thepatient's cancer has relapsed or progressed after tamoxifen treatment.In some embodiments, the patient's cancer has relapsed or progressedafter fulvestrant treatment. In some embodiments, the patient's cancerhas relapsed or progressed after aromatase inhibitor treatment. In someof these embodiments, the patient's cancer has relapsed or progressedafter multiple lines of endocrine therapy treatment.

In some embodiments, the ER⁺ cancer patient has not been treatedpreviously with endocrine therapy.

In certain embodiments, the patient is resistant to endocrine therapyother than lasofoxifene. In some embodiments, the patient has intrinsicendocrine resistance. In some embodiments, the patient has acquiredendocrine resistance. In particular embodiments, the patient isresistant to endocrine therapy due to the increased expression ofestrogen receptor. In particular embodiments, the patient is resistantto endocrine therapy due to the increased expression of co-activators ofestrogen receptor. In particular embodiments, the patient is resistantto endocrine therapy due to increased phosphorylation level and activityof estrogen receptor and its co-activators. In particular embodiments,the patient is resistant to endocrine therapy due to change of tumormicroenvironment and other host related factors. In some preferredembodiments, the patient is resistant to endocrine therapy due to genemutations in the Estrogen Receptor 1 (ESR1) gene.

In various embodiments, the patient is resistant to clinical doses ofone or more SERMs other than lasofoxifene. In some of these embodiments,the patient is resistant to clinical doses of tamoxifen. In variousembodiments, the patient is resistant to clinical doses of one or moreSERDs. In some of these embodiments, the patient is resistant toclinical doses of fulvestrant. In various embodiments, the patient isresistant to clinical doses of one or more aromatase inhibitors. Invarious embodiments, the patient is resistant to higher than clinicaldoses of one or more SERMs other than lasofoxifene. In some of theseembodiments, the patient is resistant to higher than clinical doses oftamoxifen. In various embodiments, the patient is resistant to higherthan clinical doses of one or more SERDs. In some of these embodiments,the patient is resistant to higher than clinical doses of fulvestrant.In various embodiments, the patient is resistant to higher than clinicaldoses of one or more aromatase inhibitors.

In certain embodiments, the ER⁺ cancer patient has not been demonstratedto have endocrine resistance. In some of these embodiments, the patienthas not been demonstrated to have endocrine resistance due to thelimitations of the detection methods.

In some embodiments, lasofoxifene is administered to the ER⁺ cancerpatient after completion of cancer treatment. In some of theseembodiments, lasofoxifene is administered to the patient to treat occultmicrometastasis.

6.1.3. Menopause Status

In some embodiments, the ER⁺ cancer patient is premenopausal. Inspecific embodiments, the patient is premenopausal and has locallyadvanced or metastatic ER⁺ cancer. In particular embodiments, thepatient is premenopausal and has locally advanced or metastatic ER⁺breast cancer.

In certain embodiments, the ER⁺ cancer patient is perimenopausal. Inspecific embodiments, the patient is perimenopausal and has locallyadvanced or metastatic ER⁺ cancer. In particular embodiments, thepatient is perimenopausal and has locally advanced or metastatic ER⁺breast cancer.

In typical embodiments, the ER⁺ cancer patient is postmenopausal. Inspecific embodiments, the patient is postmenopausal and has locallyadvanced or metastatic ER⁺ cancer. In particular embodiments, thepatient is postmenopausal and has locally advanced or metastatic ER⁺breast cancer.

In certain embodiments, lasofoxifene is administered to a premenopausalwoman with locally advanced or metastatic ER⁺/HER2⁻ breast cancer. Incertain embodiments, lasofoxifene is administered to a premenopausalwoman with locally advanced or metastatic ER⁺/HER2⁻ breast cancer whohas progressed while on her first hormonal treatment with a non-steroidaromatase inhibitor (AI), fulvestrant, AI in combination with a CDK4/6inhibitor, or fulvestrant in combination with a CDK4/6 inhibitor.

In certain embodiments, lasofoxifene is administered to a perimenopausalwoman with locally advanced or metastatic ER⁺/HER2⁻ breast cancer. Incertain embodiments, lasofoxifene is administered to a perimenopausalwoman with locally advanced or metastatic ER⁺/HER2⁻ breast cancer whohas progressed while on her first hormonal treatment with a non-steroidaromatase inhibitor (AI), fulvestrant, AI in combination with a CDK4/6inhibitor, or fulvestrant in combination with a CDK4/6 inhibitor.

In certain embodiments, lasofoxifene is administered to a postmenopausalwoman with locally advanced or metastatic ER⁺/HER2⁻ breast cancer. Incertain embodiments, lasofoxifene is administered to a postmenopausalwoman with locally advanced or metastatic ER⁺/HER2⁻ breast cancer whohas progressed while on her first hormonal treatment with on anon-steroid aromatase inhibitor (AI), fulvestrant, AI in combinationwith a CDK4/6 inhibitor, or fulvestrant in combination with a CDK4/6inhibitor.

6.1.4. Mutations in ESR1 Gene

In various embodiments, the patient has an ER⁺ cancer, cells of whichhave at least one mutation in the Estrogen Receptor 1 (ESR1) gene, whichencodes the Estrogen Receptor α (ERα) protein. In some embodiments, themutation leads to the ligand-independent activity of the estrogenreceptor. In some embodiments, the mutation leads to enhanced ligandstimulated activity of estrogen receptor. In some embodiments, themutation leads to resistance to endocrine therapy. In some embodiments,the mutation promotes tumor growth. In some embodiments, the mutationenhances metastatic activity of cancer. In some preferred embodiments,the mutation enhances metastatic activity of ER⁺ metastatic breastcancer.

In some embodiments, the mutation arises from a rare and undetectablepre-existing clone. In some embodiments, the mutation is acquired denovo during the course of endocrine therapy treatment. In some preferredembodiments, the mutation is acquired de novo during the course ofendocrine therapy treatment of breast cancer. In some embodiments, themutation is acquired de novo after multiple lines of endocrine therapytreatment. In some embodiments, the mutation is acquired de novo aftermultiple lines of endocrine therapy treatment of metastatic breastcancer. In various embodiments, the mutant clone expands to become amore dominant clone over the course of successive lines of endocrinetherapy.

In some embodiments, the mutation in the ESR1 gene is missense pointmutation. In some embodiments, the mutation in the ESR1 gene istruncating mutation. In some embodiments, the mutation in the ESR1 geneis gene amplification. In some embodiments, the mutation in the ESR1gene is genomic rearrangement.

In some preferred embodiments, the patient has an ER⁺ cancer that has atleast one gain of function missense mutation within the ligand bindingdomain (LBD) of the ESR1 gene. In various embodiments, at least one ofthe mutations is in an amino acid selected from D538, Y537, L536, P535,V534, S463, V392, and E380. (The amino acids are numbered according tothe ESR1 protein with the NCBI accession number NP_000116.2.)

In particular embodiments, the mutation increases the stability of theagonist conformation of Helix 12 of the ERα protein. In some of theseembodiments, the mutation increases the binding of the estrogen receptorto its co-activators. In some of these embodiments, the mutation leadsto hormone independent activity of estrogen receptor. In some of theseembodiments, the mutation leads to resistance to tamoxifen, fulvestrant,and/or aromatase inhibitors.

In certain embodiments, the mutation is in the amino acid D538. Incertain preferred embodiments, the mutation is D538G.

In certain embodiments, the mutation is in the amino acid Y537. In someof these embodiments, the mutation is Y537S, Y537N, Y537C, or Y537Q. Incertain preferred embodiments, the mutation is Y537C.

In some embodiments, the mutation is in the amino acid L536. In certainembodiments, the mutation is L536R or L536Q.

In some embodiments, the mutation is in the amino acid P535. In certainembodiments, the mutation is P535H.

In some embodiments, the mutation is in the amino acid V534. In certainembodiments, the mutation is V534E.

In some embodiments, the mutation is in the amino acid S463. In certainembodiments, the mutation is S463P.

In some embodiments, the mutation is in the amino acid V392. In certainembodiments, the mutation is V392I.

In some embodiments, the mutation is in the amino acid E380. In certainembodiments, the mutation is E380Q.

6.1.4.1. Detection of the ESR1 Gene Mutations

In various embodiments, the patient has been previously determined tohave at least one mutation in the ESR1 gene. Some embodiments of themethods described herein further include the step of detecting themutations in ESR1 gene.

In some embodiments, massively parallel next generation sequencing (NGS)is used for detecting the estrogen receptor mutations in the patient'scancer. In certain embodiments, the entire genome is sequenced. Incertain embodiments, selected gene panels of cancer-related genes aresequenced. In certain embodiments, all coding exons within a given setof genes are sequenced. In certain embodiments, known “hotspot” regionswithin a given set of genes are sequenced. However, the inherent errorrate of current next generation sequencing techniques is up to 1%,limiting the sensitivity and specificity of detection. In someembodiments, targeted sequencing is used for detecting the presence ofthe ESR1 mutations. Although targeted sequencing allows deepersequencing, it is also currently limited by the 1% error rate. In someembodiments, methods with reduced sequencing error rate are used. In aparticular embodiment, Safe-Sequencing System (Safe-SeqS) is used, whichtags each template molecule to allow for confident identification ofrare variants. See Kinde et al., Proceedings of the National Academy ofSciences 108(23): 9530-9535 (2011). In particular embodiments,ultrasensitive Duplex sequencing is used, which independently tags andsequences each of the two strands of a DNA duplex. See Schmitt et al.,Proceedings of the National Academy of Sciences 109(36): 14508-14513(2012). In some embodiments, digital droplet PCR is used, whichemulsifies DNA in thousands to millions of droplets to encapsulatesingle DNA molecules, designed with mutant specific primers. SeeVogelstein and Kinzler, Proceedings of the National Academy of Sciences96(16): 2322-2326 (1999) and Huggett et al., Clinical Chemistry 61(1):79-88 (2014).

In some embodiments, the detection of the ESR1 mutations takes place atthe initial diagnosis. In some embodiments, the detection of themutations takes place at the time of disease progression, relapse, orrecurrence. In some embodiments, the detection of the mutations takesplace at the time of disease progression. In some embodiments, thedetection of the mutations takes place at the time when the disease isstable.

In some embodiments, one or more tissue specimens are obtained fordetection of the mutations. In certain embodiments, the tissue specimenis a tumor biopsy. In certain embodiments, the tissue specimen is abiopsy of metastases. In some other embodiments, liquid biopsies areobtained for detection of the mutations. In certain embodiments, theliquid biopsy is circulating tumor cells (CTCs). In certain otherembodiments, the liquid biopsy is cell-free DNA from blood samples.

In specific embodiments, the ESR1 mutations are monitored by circulatingtumor DNA (ctDNA) analysis. In some embodiments, the ctDNA analysis isperformed throughout the course of treatment. In some of theseembodiments, the ctDNA is extracted from patient blood samples. Incertain embodiments, the ctDNA is evaluated by digital PCR analysis ofthe ESR1 mutations.

6.1.5. Estradiol Levels

In various embodiments, the patient selected for treatment based onpresence of ESR1 gene mutations is further selected based on serumestradiol level.

In certain embodiments, the serum estradiol level of the patient withthe ER⁺ cancer having an ESR1 gene mutation is at least 0.20 ng/dL, suchas at least 0.25 ng/dL, at least 0.30 ng/dL, at least 0.35 ng/dL, atleast 0.40 ng/dL, at least 0.45 ng/dL, at least 0.50 ng/dL, at least0.55 ng/dL, at least 0.60 ng/dL, at least 0.65 ng/dL, at least 0.70ng/dL, at least 0.75 ng/dL, at least 0.80 ng/dL, at least 0.85 ng/dL, atleast 0.90 ng/dL, at least 0.95 ng/dL, or at least 1.0 ng/dL.

In certain embodiments, the serum estradiol level of the patient withthe ESR1 gene mutation is about 0.20 ng/dL to about 1.0 ng/dL, such asabout 0.20 ng/dL to about 0.25 ng/dL, about 0.25 ng/dL to about 0.30ng/dL, about 0.30 ng/dL to about 0.35 ng/dL, about 0.35 ng/dL to about0.40 ng/dL, about 0.40 ng/dL to about 0.45 ng/dL, about 0.45 ng/dL toabout 0.50 ng/dL, about 0.50 ng/dL to about 0.55 ng/dL, about 0.55 ng/dLto about 0.60 ng/dL, about 0.60 ng/dL to about 0.65 ng/dL, about 0.65ng/dL to about 0.70 ng/dL, about 0.70 ng/dL to about 0.75 ng/dL, about0.75 ng/dL to about 0.80 ng/dL, about 0.80 ng/dL to about 0.85 ng/dL,about 0.85 ng/dL to about 0.90 ng/dL, about 0.90 ng/dL to about 0.95ng/dL, about 0.95 ng/dL to about 1.0 ng/dL.

6.1.6. Adjuvant Treatment

In various embodiments, lasofoxifene is administered to the patient asadjuvant treatment. In certain embodiments, lasofoxifene is administeredto the patient as adjuvant treatment alone. In certain otherembodiments, lasofoxifene is administered to the patient as adjuvanttreatment in combination with other endocrine therapies. In someembodiments, lasofoxifene is administered to the patient after theprimary treatment. In some of these embodiments, lasofoxifene isadministered to the patient after surgical removal or debulking of thecancer.

In some embodiments, lasofoxifene is administered to the patient asadjuvant therapy in combination with an aromatase inhibitor (AI). Invarious embodiments, the aromatase inhibitor is exemestane (Aromasin®),letrozole (Femara®), or anastrozole (Arimidex®).

In various embodiments, the aromatase inhibitor predisposes the patientto bone-related toxic effects. In some embodiments, the aromataseinhibitor predisposes the patient to osteoporosis. In some embodiments,the aromatase inhibitor predisposes the patient to bone loss. In someembodiments, the aromatase inhibitor predisposes the patient to bonefractures. In some embodiments, the aromatase inhibitor predisposes thepatient to bone pain.

In various embodiments, the aromatase inhibitor predisposes the patientto vulvovaginal atrophy (VVA).

In some embodiments, lasofoxifene is administered continuously duringthe administration of the aromatase inhibitor. In some otherembodiments, lasofoxifene is administered cyclically during theadministration of the aromatase inhibitor. In some embodiments,lasofoxifene and the aromatase inhibitor are administered together(simultaneously). In some other embodiments, lasofoxifene and thearomatase inhibitor are administered separately (sequentially).

In certain embodiments, the dosing regimen of lasofoxifene is differentfrom the dosing regimen of the aromatase inhibitor. In some of theseembodiments, the dosing quantity of lasofoxifene is different from thedosing quantity of the aromatase inhibitor. In some embodiments, thedosing schedule of lasofoxifene is different from the dosing schedule ofthe aromatase inhibitor. In some embodiments, the route ofadministration of lasofoxifene is different from the route ofadministration of the aromatase inhibitor.

In certain embodiments, the dosing regimen of lasofoxifene is the sameas the dosing regimen of the aromatase inhibitor. In some embodiments,the dosing quantity of lasofoxifene is the same as the dosing quantityof the aromatase inhibitor. In some embodiments, the dosing schedule oflasofoxifene is the same as the dosing schedule of the aromataseinhibitor. In some embodiments, the route of administration oflasofoxifene is the same as the route of administration of the aromataseinhibitor.

In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for one year. Insome embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for two years. Insome embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for three years.In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for four years.In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for five years.In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for six years. Insome embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for seven years.In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for eight years.In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for nine years.In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to the patient for ten years. Insome other embodiments, lasofoxifene is administered as adjuvant therapyin combination with an aromatase inhibitor to the patient for more thanten years. In certain embodiments, lasofoxifene is administered asadjuvant therapy in combination with an aromatase inhibitor until thepatient's cancer progresses on therapy.

In some embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to increase the disease-freesurvival of the breast cancer patient. In some embodiments, lasofoxifeneis administered as adjuvant therapy in combination with an aromataseinhibitor to decrease the incidence of contralateral breast cancer. Insome embodiments, lasofoxifene is administered as adjuvant therapy incombination with an aromatase inhibitor to prevent the recurrence orprogression of the cancer.

6.2. LASOFOXIFENE

In various embodiments, the selected patient is treated with aneffective amount of lasofoxifene, a pharmaceutically acceptable saltthereof, or a prodrug thereof. In some preferred embodiments,lasofoxifene is administered to the selected patient as lasofoxifenetartrate.

The term “pharmaceutically acceptable salt” refers to non-toxicpharmaceutically acceptable salts. See Gould, International Journal ofPharmaceutics 33: 201-217 (1986) and Berge et al., Journal ofPharmaceutical Sciences 66(1): 1-19 (1977). Other salts well known tothose in the art may, however, be used. Representative organic orinorganic acids include, but are not limited to, hydrochloric,hydrobromic, hydriodic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic,tartaric, citric, benzoic, mandelic, methanesulfonic,hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,salicylic, saccharinic or trifluoroacetic acid. Representative organicor inorganic bases include, but are not limited to, basic or cationicsalts such as benzathine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine, procaine, aluminum, calcium, lithium,magnesium, potassium, sodium and zinc.

Embodiments also include prodrugs of the compounds disclosed herein. Ingeneral, such prodrugs will be functional derivatives of the compoundswhich are readily convertible in vivo into the required compound. Thus,in the methods of treatment of the present invention, the term“administering” shall encompass the treatment of the various disordersdescribed with the compound specifically disclosed or with a compoundwhich may not be specifically disclosed, but which converts to thespecified compound in vivo after administration to the subject.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs”,H. Bundgaard, Elsevier, 1985.

Some of the crystalline forms for the compounds may exist as polymorphsand as such are intended to be included in the present invention. Inaddition, some of the compounds may form solvates with water (i.e.,hydrates) or common organic solvents, and such solvates are intended tobe encompassed by some embodiments.

Where the processes for the preparation of the compounds as disclosedherein give rise to mixtures of stereoisomers, these isomers may beseparated by conventional techniques such as preparative chromatography.The compounds may be prepared in racemic form or as individualenantiomers or diastereomers by either stereospecific synthesis or byresolution. The compounds may, for example, be resolved into theircomponent enantiomers or diastereomers by standard techniques, such asthe formation of stereoisomeric pairs by salt formation with anoptically active base, followed by fractional crystallization andregeneration of the free acid. The compounds may also be resolved byformation of stereoisomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral HPLC column.It is to be understood that all stereoisomers, racemic mixtures,diastereomers, cis-trans isomers, and enantiomers thereof areencompassed by some embodiments.

6.3. PHARMACEUTICAL COMPOSITIONS

Methods for treatment of estrogen receptor positive (ER⁺) cancersinclude administering a therapeutically effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof. The lasofoxifene, the pharmaceutically acceptable salt, or theprodrug of the invention can be formulated in pharmaceuticalcompositions. In addition to lasofoxifene, a pharmaceutically acceptablesalt thereof, or a prodrug thereof, the composition further comprises apharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialcan depend on the route of administration, e.g. oral, intravenous,transdermal, vaginal topical, or vaginal ring.

Pharmaceutical compositions for oral administration can be in tablet,capsule, powder or liquid form. A tablet can include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal oil,vegetable oil, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol can also beincluded.

For parenteral administration, the lasofoxifene will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives can be included, as required.

Pharmaceutical compositions for vaginal topical administration can be inthe form of ointment, cream, gel or lotion. The pharmaceuticalcompositions for vaginal topical administration often include water,alcohol, animal oil, vegetable oil, mineral oil or synthetic oil.Hydrocarbon (paraffin), wool fat, beeswax, macrogols, emulsifying wax orcetrimide can also be included.

A composition can be administered alone or in combination with othertreatments, either simultaneously or sequentially, dependent upon thecondition to be treated.

6.4. TREATMENT REGIMENS

In the methods of administering an effective amount of lasofoxifene inthe form of a pharmaceutical composition as described above fortreatment of ER⁺ cancer, the terms “treatment”, “treating”, and the likeare used herein to generally mean obtaining a desired pharmacologicand/or physiologic effect. The effect may be prophylactic, in terms ofcompletely or partially preventing a disease, condition, or symptomsthereof, and/or may be therapeutic in terms of a partial or completecure for a disease or condition and/or adverse effect, such as asymptom, attributable to the disease or condition. “Treatment” as usedherein covers any treatment of a disease or condition of a mammal,particularly a human, and includes: (a) preventing the disease orcondition from occurring in a subject which may be predisposed to thedisease or condition but has not yet been diagnosed as having it; (b)inhibiting the disease or condition (e.g., arresting its development);or (c) relieving the disease or condition (e.g., causing regression ofthe disease or condition, providing improvement in one or moresymptoms). Improvements in any conditions can be readily assessedaccording to standard methods and techniques known in the art. Thepopulation of subjects treated by the method of the disease includessubjects suffering from the undesirable condition or disease, as well assubjects at risk for development of the condition or disease.

The term “effective amount” means a dose that produces the desiredeffect for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques. See Lloyd, The Art, Science andTechnology of Pharmaceutical Compounding (1999).

6.4.1. Routes of Administration

In various embodiments, lasofoxifene is administered by oral,intravenous, transdermal, vaginal topical, or vaginal ringadministration.

In some embodiments, lasofoxifene is administered to the patient by oraladministration. In certain embodiments, lasofoxifene is administered atabout 0.5 mg/day per os to about 10 mg/day per os, such as about 0.5mg/day per os to about 5 mg/day per os, about 0.5 mg/day per os to about5 mg/day per os, about 1 mg/day per os to about 5 mg/day per os, about 2mg/day per os to about 5 mg/day per os, about 3 mg/day per os to about 5mg/day per os, about 4 mg/day per os to about 5 mg/day per os, about 0.5mg/day per os to about 4 mg/day per os, about 1 mg/day per os to about 4mg/day per os, about 2 mg/day per os to about 4 mg/day per os, about 3mg/day per os to about 4 mg/day per os, about 0.5 mg/day per os to about3 mg/day per os, about 1 mg/day per os to about 3 mg/day per os, about 2mg/day per os to about 3 mg/day per os, about 0.5 mg/day per os to about2 mg/day per os, about 1 mg/day per os to about 2 mg/day per os, orabout 0.5 mg/day per os to about 1 mg/day per os. In some embodiments,lasofoxifene is administered at about 0.5 mg/day per os. In someembodiments, lasofoxifene is administered at about 1 mg/day per os. Insome embodiments, lasofoxifene is administered at about 1.5 mg/day peros. In some embodiments, lasofoxifene is administered at about 2 mg/dayper os. In some embodiments, lasofoxifene is administered at about 2.5mg/day per os. In some embodiments, lasofoxifene is administered atabout 3 mg/day per os. In some embodiments, lasofoxifene is administeredat about 3.5 mg/day per os. In some embodiments, lasofoxifene isadministered at about 4 mg/day per os. In some embodiments, lasofoxifeneis administered at about 4.5 mg/day per os. In some embodiments,lasofoxifene is administered at about 5 mg/day per os. In someembodiments, lasofoxifene is administered at about 6 mg/day per os. Insome embodiments, lasofoxifene is administered at about 7 mg/day per os.In some embodiments, lasofoxifene is administered at about 8 mg/day peros. In some embodiments, lasofoxifene is administered at about 9 mg/dayper os. In some embodiments, lasofoxifene is administered at about 10mg/day per os. In some other embodiments, lasofoxifene is administeredat more than 10 mg/day per os.

In certain embodiments, when lasofoxifene is administered to patientwhose cancer has not acquired endocrine resistance, lasofoxifene can beadministered at less than 0.5 mg/day per os for prevention of endocrineresistance. In certain embodiments, when lasofoxifene is administered tocancer patient as adjuvant treatment, lasofoxifene can be administeredat less than 0.5 mg/day per os for prevention of endocrine resistance.

In certain embodiments, lasofoxifene is administered once every day. Incertain embodiments, lasofoxifene is administered once every two days.In certain embodiments, lasofoxifene is administered once every threedays. In certain embodiments, lasofoxifene is administered once everyfour days. In certain embodiments, lasofoxifene is administered onceevery five days. In certain embodiments, lasofoxifene is administeredonce every six days. In certain embodiments, lasofoxifene isadministered once every week. In certain embodiments, lasofoxifene isadministered once every two weeks. In certain embodiments, lasofoxifeneis administered once every three weeks. In certain embodiments,lasofoxifene is administered once every month.

In some embodiments, lasofoxifene is administered to the patient byvaginal ring administration. In some of these embodiments, lasofoxifeneis administered once every two weeks. In some of these embodiments,lasofoxifene is administered once every three weeks. In some of theseembodiments, lasofoxifene is administered once every month. In some ofthese embodiments, lasofoxifene is administered once every two months.In some of these embodiments, lasofoxifene is administered once everythree months. In some of these embodiments, lasofoxifene is administeredonce every four months.

In some embodiments, lasofoxifene is administered to ER⁺ cancer patientfor one year. In some embodiments, lasofoxifene is administered to thepatient for two years. In some embodiments, lasofoxifene is administeredto the patient for three years. In some embodiments, lasofoxifene isadministered to the patient for four years. In some embodiments,lasofoxifene is administered to the patient for five years. In someother embodiments, lasofoxifene is administered to the patient for morethan five years. In certain embodiments, lasofoxifene is administered tothe patient until the patient's cancer progresses on therapy.

6.4.2. Combination Therapy

In various embodiments, lasofoxifene is administered either alone or incombination with other therapies. In certain embodiments, lasofoxifeneis administered in combination with at least one other therapy. In someembodiments, lasofoxifene and other therapies are administered together(simultaneously). In some other embodiments, lasofoxifene and othertherapies are administered at different times (sequentially).

In particular embodiments, the additional therapy that the patient istreated with is endocrine therapy. In various embodiments, the patientis treated with at least one line of additional endocrine therapy. Insome embodiments, the patient is treated with one line of additionalendocrine therapy. In some other embodiments, the patient is treatedwith multiple lines of additional endocrine therapy.

In some embodiments, the patient is treated with the additionalendocrine therapy at the original doses. In some other embodiments, thepatient is treated with the additional endocrine therapy at doses higherthan original doses. In certain embodiments, the patient is treated withthe additional endocrine therapy at doses lower than original doses.

In certain embodiments, the additional endocrine therapy is treatmentwith a selective ER modulator (SERM) other than lasofoxifene. In some ofthese embodiments, the selective ER modulator is selected fromtamoxifen, raloxifene, bazedoxifene, toremifene, and ospermifene. Incertain embodiments, the selective ER modulator is tamoxifen.

In certain embodiments, the additional endocrine therapy is treatmentwith a selective ER degrader (SERD). In some of these embodiments, theselective ER degrader is selected from fulvestrant, RAD1901, ARN-810(GDC-0810), and AZD9496. In certain embodiments, the selective ERdegrader is fulvestrant.

In certain embodiments, the additional endocrine therapy is treatmentwith an aromatase inhibitor. In some of these embodiments, the aromataseinhibitor is selected from exemestane (Aromasin®), letrozole (Femara®),and anastrozole (Arimidex®).

In various embodiments, the additional therapy is administration to thepatient of an effective amount of a cell cycle inhibitor. In certainembodiments, the additional therapy is administration of an effectiveamount of cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. In someembodiments, the additional therapy is a CDK4/6 inhibitor selected fromthe group of palbociclib, abemaciclib, and ribociclib.

In some embodiments, the additional therapy is administration to thepatient of an inhibitor of a pathway that cross-talks with and activatesthe ER transcriptional activity. In certain embodiments, the additionaltherapy is a mammalian target of rapamycin (mTOR) inhibitor. In specificembodiments, the mTOR inhibitor is Everolimus. In some of theseembodiments, lasofoxifene in combination with Everolimus is administeredto a postmenopausal woman with locally advanced or metastatic breastcancer who has progressed on a non-steroidal AI and/or fulvestranteither as monotherapy or in combination with a CDK4/6 inhibitor. Invarious embodiments, the additional therapy is a phosphoinositide3-kinase (PI3K) inhibitor or a heat shock protein 90 (HSP90) inhibitor.

In various embodiments, the additional therapy is administration to thepatient of an effective amount of a growth factor inhibitor. In certainembodiments, the additional therapy is a human epidermal growth factorreceptor 2 (HER2) inhibitor. In some embodiments, the HER2 inhibitor istrastuzumab (Herceptin®). In some other embodiments, the HER2 inhibitoris ado-trastuzumab emtansine (Kadcyla®).

In some embodiments, the additional therapy is administering to thepatient an effective amount of a histone deacetylase (HDAC) inhibitor.In various embodiments, the HDAC inhibitor is vorinostat (Zolinza®),romidepsin (Istodax®), chidamide (Epidaza®), panobinostat (Farydak®),belinostat (Beleodaq®, PXD101), valproic acid (Depakote®, Depakene®,Stavzor®), mocetinostat (MGCD0103), abexinostat (PCI-24781), entinostat(MS-275), pracinostat (SB939), resminostat (4SC-201), givinostat(ITF2357), quisinostat (JNJ-26481585), kevetrin, CUDC-101, AR-42,tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745, rocilinostat(ACY-1215), or sulforaphane. In certain embodiments, the HDAC inhibitoris entinostat (MS-275) with the proviso that the patient is not treatedwith a HER2 inhibitor. In certain other embodiments, the HDAC inhibitoris vorinostat (Zolinza®). In yet certain other embodiments, the HDACinhibitor is romidepsin (Istodax®).

In some embodiments, the additional therapy is administering to thepatient an effective amount of a checkpoint inhibitor. In certainembodiments, the checkpoint inhibitor is an antibody. In some of theseembodiments, the checkpoint inhibitor is an antibody specific forprogrammed cell death protein 1 (PD-1), programmed death-ligand 1(PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Insome embodiments, the PD-1 antibody is pembrolizumab (Keytruda®) ornivolumab (Opdivo®). In some embodiments, the CTLA-4 antibody isipilimumab (Yervoy®).

In certain embodiments, the additional therapy is administering to thepatient an effective amount of cancer vaccine.

In some embodiments, the additional therapy is administering to thepatient an effective amount of denosumab.

In some embodiments, the additional therapy is administering to thepatient an effective amount of a serotonin-norepinephrine reuptakeinhibitor (SNRI), a selective serotonin reuptake inhibitor (SSRI), orgabapentin. In certain embodiments, the SNRI is venlafaxine (Effexor®).

6.4.3. Clinical Endpoints 6.4.3.1. Primary Clinical Endpoints

In various embodiments, the method comprises administering an amount oflasofoxifene effective to increase the disease-free survival of the ER⁺cancer patient. In some embodiments, the method comprises administeringlasofoxifene in an amount effective to reduce recurrence of ER⁺ cancer.In some embodiments, the method comprises administering lasofoxifene inan amount effective to increase time to recurrence of ER⁺ cancer. Insome embodiments, the method comprises administering lasofoxifene in anamount effective to reduce metastasis of ER⁺ cancer. In someembodiments, the method comprises administering lasofoxifene in anamount effective to increase duration of progression-free survival ofthe ER⁺ cancer patient.

In various embodiments, the method increases the disease-free survivalof the ER⁺ breast cancer patient. In certain embodiments, the methodreduces recurrence of ER⁺ breast cancer. In certain embodiments, themethod increases time to recurrence of ER⁺ breast cancer. In certainembodiments, the method reduces metastasis of ER⁺ breast cancer to bone.In certain embodiments, the method reduces metastasis of ER⁺ breastcancer to tissues other than bone. In certain embodiments, the methodincreases duration of progression-free survival of the ER⁺ breast cancerpatient.

In various embodiments, the method increases the disease-free survivalin ER⁺ cancer patient with endocrine resistance. In some embodiments,the method reduces recurrence of cancer in patient with endocrineresistance. In some embodiments, the method increases time to recurrenceof cancer in patient with endocrine resistance. In some embodiments, themethod reduces metastasis of cancer in patient with endocrineresistance. In some embodiments, the method increases duration ofprogression-free survival in ER⁺ cancer patient with endocrineresistance.

In some preferred embodiments, the method increases disease-freesurvival, reduces recurrence, increases time to recurrence, reducesmetastasis, and/or increases duration of progression-free survival inpatients with ER⁺ locally advanced or metastatic breast cancer that hasdeveloped endocrine resistance. In particular embodiments, the breastcancer has developed endocrine resistance by acquiring one or more ofthe ESR1 mutations discussed herein. In some embodiments, the methodreduces the selective pressure and prevents the expansion of theendocrine resistant clones in ER⁺ locally advanced or metastatic breastcancer during treatment.

6.4.3.2. Secondary Clinical Endpoints

In some embodiments, the method is effective to prevent fracture andbone loss in women who are concurrently being treated with one or moredrugs causing or predisposing to osteoporosis.

In some embodiments, the method is effective to decrease vaginal pH,increase vaginal lubrication, and/or improve vaginal cell maturationindex in women who are concurrently being treated with one or more drugscausing or predisposing to vulvovaginal atrophy (VVA).

In some embodiments, the method reduces one or more symptoms of sexualdysfunction in women who are concurrently being treated with one or moredrugs causing or predisposing to sexual dysfunction.

In some embodiments, the method treats hot flashes in women who areconcurrently being treated with one or more drugs causing orpredisposing to hot flashes.

In some embodiments, the method increases one or more quality of lifemeasures selected from joint ache, urogenital symptoms, bone loss, andbone fractures.

6.5. FURTHER EMBODIMENTS

Further embodiments are provided in the following numbered embodiments.

1. A method of treating locally advanced or metastatic breast cancer inwomen, comprising:

a) selecting for treatment a patient who has been diagnosed withestrogen receptor positive (ER+) locally advanced or metastatic breastcancer; and

b) administering to the selected patient an effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof.

2. The method of embodiment 1, wherein the patient has previously beentreated with one or more lines of endocrine therapy.3. The method of embodiment 2, wherein the patient has previously beentreated with a plurality of lines of endocrine therapy.4. The method of embodiment 2 or embodiment 3, wherein the endocrinetherapy that the patient has previously been treated with is a selectiveER modulator (SERM).5. The method of embodiment 4, wherein the SERM is tamoxifen,raloxifene, bazedoxifene, toremifene, or ospemifene.6. The method of embodiment 2 or embodiment 3, wherein the endocrinetherapy that the patient has previously been treated with is a selectiveER degrader (SERD).7. The method of embodiment 6, wherein the SERD is fulvestrant, RAD1901,ARN-810 (GDC-0810), or AZD9496.8. The method of embodiment 2 or embodiment 3, wherein the endocrinetherapy that the patient has previously been treated with is anaromatase inhibitor.9. The method of embodiment 8, wherein the aromatase inhibitor isexemestane (Aromasin®), letrozole (Femara®), or anastrozole (Arimidex®).10. The method of any one of embodiments 2 to 9, wherein the patient hasdisease progression after endocrine therapy.11. The method of any one of embodiments 1 to 10, wherein the patient'slocally advanced or metastatic cancer is resistant to endocrine therapyother than lasofoxifene.12. The method of any one of embodiments 1 to 11, wherein the patient'slocally advanced or metastatic cancer has at least one gain of functionmissense mutation within the ligand binding domain (LBD) of the EstrogenReceptor 1 (ESR1) gene.13. The method of embodiment 12, wherein the patient has previously beendetermined to have at least one gain of function missense mutationwithin the ligand binding domain (LBD) of the Estrogen Receptor 1 (ESR1)gene.14. The method of embodiment 13, further comprising the earlier step of:

determining that the patient has at least one gain of function missensemutation within the ligand binding domain (LBD) of the Estrogen Receptor1 (ESR1) gene.

15. The method of any one of embodiments 12 to 14, wherein the at leastone of gain of function missense mutation is in any one of amino acidsD538, Y537, L536, P535, V534, S463, V392, and E380.16. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid D538.17. The method of embodiment 16, wherein the mutation is D538G.18. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid Y537.19. The method of embodiment 18, wherein the mutation is Y537S, Y537N,Y537C, or Y537Q.20. The method of embodiment 19, wherein the mutation is Y537C.21. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid L536.22. The method of embodiment 21, wherein the mutation is L536R or L536Q.23. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid P535.24. The method of embodiment 23, wherein the mutation is P535H.25. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid V534.26. The method of embodiment 25, wherein the mutation is V534E.27. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid S463.28. The method of embodiment 27, wherein the mutation is S463P.29. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid V392.30. The method of embodiment 29, wherein the mutation is V392I.31. The method of embodiment 15, wherein the at least one gain offunction missense mutation is in the amino acid E380.32. The method of embodiment 31, wherein the mutation is E380Q.33. The method of any one of embodiments 12 to 32, wherein the serumestradiol level of the patient is at least 0.35 ng/dL.34. The method of any one of embodiments 12 to 32, wherein the serumestradiol level of the patient is about 0.30 ng/dL to about 0.35 ng/dL.35. The method of any one of embodiments 12 to 32, wherein the serumestradiol level of the patient is about 0.25 ng/dL to about 0.30 ng/dL.36. The method of any one of embodiments 1 to 35, wherein lasofoxifeneis administered as lasofoxifene tartrate.37. The method of any one of embodiments 1 to 36, wherein lasofoxifeneis administered by oral, intravenous, transdermal, vaginal topical, orvaginal ring administration.38. The method of embodiment 37, wherein lasofoxifene is administered byoral administration.39. The method of embodiment 38, wherein lasofoxifene is administered atabout 0.5 mg/day per os to about 10 mg/day per os.40. The method of embodiment 39, wherein lasofoxifene is administered atabout 0.5 mg/day per os to about 5 mg/day per os.41. The method of embodiment 40, wherein lasofoxifene is administered atabout 1 mg/day per os to about 5 mg/day per os.42. The method of embodiment 40, wherein lasofoxifene is administered at1 mg/day per os.43. The method of embodiment 40, wherein lasofoxifene is administered at5 mg/day per os.44. The method of any one of embodiments 1 to 43, wherein lasofoxifeneis administered once every day, once every two days, once every threedays, once every four days, once every five days, once every six days,once every week, once every two weeks, once every three weeks, or onceevery month.45. The method of any one of embodiments 1 to 44, further comprisingtreating said patient with at least one additional endocrine therapy.46. The method of embodiment 45, wherein said patient is treated withthe additional endocrine therapy at original doses.47. The method of embodiment 45, wherein said patient is treated withthe additional endocrine therapy at doses higher than original doses.48. The method of any one of embodiments 45 to 47, wherein theadditional endocrine therapy is treatment with a selective ER modulator(SERM) other than lasofoxifene.49. The method of any one of embodiments 45 to 47, wherein theadditional endocrine therapy is treatment with a selective ER degrader(SERD).50. The method of any one of embodiments 45 to 47, wherein theadditional endocrine therapy is treatment with an aromatase inhibitor.51. The method of any one of embodiments 1 to 44, further comprisingadministering to said patient an effective amount of cyclin-dependentkinase 4/6 (CDK4/6) inhibitor.52. The method of embodiment 51, wherein said CDK4/6 inhibitor ispalbociclib, abemaciclib, or ribociclib.53. The method of any one of embodiments 1 to 44, further comprisingadministering to said patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor.54. The method of embodiment 53, wherein said mTOR inhibitor isEverolimus.55. The method of any one of embodiments 1 to 44, further comprisingadministering to said patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor.56. The method of any one of embodiments 1 to 44, further comprisingadministering to said patient an effective amount of human epidermalgrowth factor receptor 2 (HER2) inhibitor.57. The method of embodiment 56, wherein said HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®).58. The method of any one of embodiments 1 to 44, further comprisingadministering to said patient an effective amount of a histonedeacetylase (HDAC) inhibitor.59. The method of embodiment 58, wherein said HDAC inhibitor isvorinostat (Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®),panobinostat (Farydak®), belinostat (Beleodaq®, PXD101), valproic acid(Depakote®, Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat(PCI-24781), entinostat (MS-275), pracinostat (SB939), resminostat(4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin,CUDC-101, AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745,rocilinostat (ACY-1215), or sulforaphane.60. The method of any one of embodiments 1 to 44, further comprisingadministering to said patient an effective amount of a checkpointinhibitor.61. The method of embodiment 60, wherein said checkpoint inhibitor is anantibody specific for programmed cell death protein 1 (PD-1), programmeddeath-ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4(CTLA-4).62. The method of embodiment 61, wherein said PD-1 antibody ispembrolizumab (Keytruda®) or nivolumab (Opdivo®).63. The method of embodiment 61, wherein said CTLA-4 antibody isipilimumab (Yervoy®).64. The method of any one of embodiments 1 to 44, further comprisingadministering to said patient an effective amount of cancer vaccine.65. The method of any one of embodiments 1 to 64, wherein the patient ispremenopausal.66. The method of embodiment 65, wherein the patient has locallyadvanced or metastatic ER+/HER2− breast cancer.67. The method of embodiment 65, wherein the patient has progressed onher first hormonal treatment while on a non-steroid aromatase inhibitor(AI), fulvestrant, AI in combination with a CDK4/6 inhibitor, orfulvestrant in combination with a CDK4/6 inhibitor.68. The method of any one of embodiments 1 to 64, wherein the patient isperimenopausal.69. The method of embodiment 68, wherein the patient has locallyadvanced or metastatic ER+/HER2− breast cancer.70. The method of embodiment 69, wherein the patient has progressed onher first hormonal treatment while on a non-steroid aromatase inhibitor(AI), fulvestrant, AI in combination with a CDK4/6 inhibitor, orfulvestrant in combination with a CDK4/6 inhibitor.71. The method of any one of embodiments 1 to 64, wherein the patient ispostmenopausal.72. The method of embodiment 71, wherein the patient has locallyadvanced or metastatic ER+/HER2− breast cancer.73. The method of embodiment 72, wherein the patient has progressed onher first hormonal treatment while on a non-steroid aromatase inhibitor(AI), fulvestrant, AI in combination with a CDK4/6 inhibitor, orfulvestrant in combination with a CDK4/6 inhibitor.74. A method of treating primary breast cancer in women, comprising:

a) selecting for treatment a patient who has been diagnosed withestrogen receptor positive (ER+) primary breast cancer; and

b) administering to the selected patient an effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof.

75. The method of embodiment 74, wherein lasofoxifene is administered aslasofoxifene tartrate.76. The method of embodiment 74 or embodiment 75, wherein lasofoxifeneis administered by oral, intravenous, transdermal, vaginal topical, orvaginal ring administration.77. The method of embodiment 76, wherein lasofoxifene is administered byoral administration.78. The method of embodiment 77, wherein lasofoxifene is administered atabout 0.5 mg/day per os to about 10 mg/day per os.79. The method of embodiment 78, wherein lasofoxifene is administered atabout 0.5 mg/day per os to about 5 mg/day per os.80. The method of embodiment 79, wherein lasofoxifene is administered atabout 1 mg/day per os to about 5 mg/day per os.81. The method of embodiment 79, wherein lasofoxifene is administered at1 mg/day per os.82. The method of embodiment 79, wherein lasofoxifene is administered at5 mg/day per os.83. The method of any one of embodiments 74 to 82, wherein lasofoxifeneis administered once every day, once every two days, once every threedays, once every four days, once every five days, once every six days,once every week, once every two weeks, once every three weeks, or onceevery month.84. The method of any one of embodiments 74 to 83, further comprisingtreating said patient with at least one additional endocrine therapy.85. The method of embodiment 84, wherein said patient is treated withthe additional endocrine therapy at original doses.86. The method of embodiment 84, wherein said patient is treated withthe additional endocrine therapy at doses higher than original doses.87. The method of any one of embodiments 84 to 86, wherein theadditional endocrine therapy is treatment with a selective ER modulator(SERM) other than lasofoxifene.88. The method of any one of embodiments 84 to 86, wherein theadditional endocrine therapy is treatment with a selective ER degrader(SERD).89. The method of any one of embodiments 84 to 86, wherein theadditional endocrine therapy is treatment with an aromatase inhibitor.90. The method of any one of embodiments 74 to 83, further comprisingadministering to said patient an effective amount of cyclin-dependentkinase 4/6 (CDK4/6) inhibitor.91. The method of embodiment 90, wherein said CDK4/6 inhibitor ispalbociclib, abemaciclib, or ribociclib.92. The method of any one of embodiments 74 to 83, further comprisingadministering to said patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor.93. The method of embodiment 92, wherein said mTOR inhibitor isEverolimus.94. The method of any one of embodiments 74 to 83, further comprisingadministering to said patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor.95. The method of any one of embodiments 74 to 83, further comprisingadministering to said patient an effective amount of human epidermalgrowth factor receptor 2 (HER2) inhibitor.96. The method of embodiment 95, wherein said HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®).97. The method of any one of embodiments 74 to 83, further comprisingadministering to said patient an effective amount of a histonedeacetylase (HDAC) inhibitor.98. The method of embodiment 97, wherein said HDAC inhibitor isvorinostat (Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®),panobinostat (Farydak®), belinostat (Beleodaq®, PXD101), valproic acid(Depakote®, Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat(PCI-24781), entinostat (MS-275), pracinostat (SB939), resminostat(4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin,CUDC-101, AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745,rocilinostat (ACY-1215), or sulforaphane.99. The method of any one of embodiments 74 to 83, further comprisingadministering to said patient an effective amount of checkpointinhibitor.100. The method of embodiment 99, wherein said checkpoint inhibitor isan antibody specific for programmed cell death protein 1 (PD-1),programmed death-ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4).101. The method of embodiment 100, wherein said PD-1 antibody ispembrolizumab (Keytruda®) or nivolumab (Opdivo®).102. The method of embodiment 100, wherein said CTLA-4 antibody isipilimumab (Yervoy®).103. The method of any one of embodiments 74 to 83, further comprisingadministering to said patient an effective amount of cancer vaccine.104. The method of any one of embodiments 74 to 103, wherein the patientis premenopausal.105. The method of any one of embodiments 74 to 103, wherein the patientis perimenopausal.106. The method of any one of embodiments 74 to 103, wherein the patientis postmenopausal.107. A method of adjuvant therapy of estrogen receptor positive (ER+)breast cancer, comprising:

administering to a patient who has received primary treatment for ER+breast cancer an effective amount of lasofoxifene, a pharmaceuticallyacceptable salt thereof, or a prodrug thereof, in combination with anaromatase inhibitor.

108. The method of embodiment 107, wherein lasofoxifene is administeredcontinuously during the administration of the aromatase inhibitor.109. The method of embodiment 107, wherein lasofoxifene is administeredcyclically during the administration of the aromatase inhibitor.110. The method of any one of embodiments 107 to 109, wherein the dosingregimen of lasofoxifene is different from the dosing regimen of thearomatase inhibitor.111. The method of any one of embodiments 107 to 110, whereinlasofoxifene is administered as lasofoxifene tartrate.112. The method of any one of embodiments 107 to 111, wherein thearomatase inhibitor is exemestane (Aromasin®), letrozole (Femara®), oranastrozole (Arimidex®).113. The method of any one of embodiments 107 to 112, whereinlasofoxifene is administered by oral, intravenous, transdermal, vaginaltopical, or vaginal ring administration.114. The method of embodiment 113, wherein lasofoxifene is administeredby oral administration.115. The method of embodiment 114, wherein lasofoxifene is administeredat about 0.5 mg/day per os to about 10 mg/day per os.116. The method of embodiment 115, wherein lasofoxifene is administeredat about 0.5 mg/day per os to about 5 mg/day per os.117. The method of embodiment 116, wherein lasofoxifene is administeredat about 1 mg/day per os to about 5 mg/day per os.118. The method of embodiment 116, wherein lasofoxifene is administeredat 1 mg/day per OS.119. The method of embodiment 116, wherein lasofoxifene is administeredat 5 mg/day per OS.120. The method of any one of embodiments 107 to 119, whereinlasofoxifene is administered once every day, once every two days, onceevery three days, once every four days, once every five days, once everysix days, once every week, once every two weeks, once every three weeks,or once every month.121. The method of any one of embodiments 107 to 120, further comprisingtreating said patient with an additional endocrine therapy.122. The method of embodiment 121, wherein the additional endocrinetherapy is treatment with a selective ER degrader (SERD).123. The method of any one of embodiments 107 to 120, further comprisingadministering to said patient an effective amount of cyclin-dependentkinase 4/6 (CDK4/6) inhibitor.124. The method of embodiment 123, wherein said CDK4/6 inhibitor ispalbociclib, abemaciclib, or ribociclib.125. The method of any one of embodiments 107 to 120, further comprisingadministering to said patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor.126. The method of embodiment 125, wherein said mTOR inhibitor isEverolimus.127. The method of any one of embodiments 107 to 120, further comprisingadministering to said patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor.128. The method of any one of embodiments 107 to 120, further comprisingadministering to said patient an effective amount of human epidermalgrowth factor receptor 2 (HER2) inhibitor.129. The method of embodiment 128, wherein said HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®).130. The method of any one of embodiments 107 to 120, further comprisingadministering to said patient an effective amount of a histonedeacetylase (HDAC) inhibitor.131. The method of embodiment 130, wherein said HDAC inhibitor isvorinostat (Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®),panobinostat (Farydak®), belinostat (Beleodaq®, PXD101), valproic acid(Depakote®, Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat(PCI-24781), entinostat (MS-275), pracinostat (SB939), resminostat(4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin,CUDC-101, AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745,rocilinostat (ACY-1215), or sulforaphane.132. The method of any one of embodiments 107 to 120, further comprisingadministering to said patient an effective amount of checkpointinhibitor.133. The method of embodiment 132, wherein said checkpoint inhibitor isan antibody specific for programmed cell death protein 1 (PD-1),programmed death-ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4).134. The method of embodiment 133, wherein said PD-1 antibody ispembrolizumab (Keytruda®) or nivolumab (Opdivo®).135. The method of embodiment 133, wherein said CTLA-4 antibody isipilimumab (Yervoy®).136. The method of any one of embodiments 107 to 120, further comprisingadministering to said patient an effective amount of cancer vaccine.137. The method of any one of embodiments 107 to 136, whereinlasofoxifene is administered in an amount and on a schedule sufficientto improve bone mass.138. The method of any one of embodiments 107 to 136, whereinlasofoxifene is administered in an amount and on a schedule sufficientto improve symptoms of VVA.139. The method of any one of embodiments 107 to 138, wherein thepatient is premenopausal.140. The method of any one of embodiments 107 to 138, wherein thepatient is perimenopausal.141. The method of any one of embodiments 107 to 138, wherein thepatient is postmenopausal.142. A method of treating cancers other than breast cancer in women,comprising:

a) selecting for treatment a patient who has been diagnosed withestrogen receptor positive (ER+) cancer, other than breast cancer, andhas at least one gain of function mutations in the Estrogen Receptor 1(ESR1) gene; and

b) administering to the selected patient an effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof.

143. The method of embodiment 142, wherein the patient has beendiagnosed with ER+ ovarian cancer.

144. The method of embodiment 142, wherein the patient has beendiagnosed with ER+ lung cancer.

145. The method of any one of embodiments 142 to 144, whereinlasofoxifene is administered as lasofoxifene tartrate.146. The method of any one of embodiments 142 to 145, whereinlasofoxifene is administered by oral, intravenous, transdermal, vaginaltopical, or vaginal ring administration.147. The method of embodiment 146, wherein lasofoxifene is administeredby oral administration.148. The method of embodiment 147, wherein lasofoxifene is administeredat about 0.5 mg/day per os to about 10 mg/day per os.149. The method of embodiment 148, wherein lasofoxifene is administeredat about 0.5 mg/day per os to about 5 mg/day per os.150. The method of embodiment 149, wherein lasofoxifene is administeredat about 1 mg/day per os to about 5 mg/day per os.151. The method of embodiment 149, wherein lasofoxifene is administeredat 1 mg/day per OS.152. The method of embodiment 149, wherein lasofoxifene is administeredat 5 mg/day per OS.153. The method of any one of embodiments 142 to 152, whereinlasofoxifene is administered once every day, once every two days, onceevery three days, once every four days, once every five days, once everysix days, once every week, once every two weeks, once every three weeks,or once every month.154. The method of any one of embodiments 142 to 153, further comprisingtreating said patient with at least one additional endocrine therapy.155. The method of embodiment 154, wherein said patient is treated withthe additional endocrine therapy at original doses.156. The method of embodiment 154, wherein said patient is treated withthe additional endocrine therapy at doses higher than original doses.157. The method of any one of embodiments 154 to 156, wherein theadditional endocrine therapy is treatment with a selective ER modulator(SERM) other than lasofoxifene.158. The method of any one of embodiments 154 to 156, wherein theadditional endocrine therapy is treatment with a selective ER degrader(SERD).159. The method of any one of embodiments 154 to 156, wherein theadditional endocrine therapy is treatment with an aromatase inhibitor.160. The method of any one of embodiments 142 to 153, further comprisingadministering to said patient an effective amount of cyclin-dependentkinase 4/6 (CDK4/6) inhibitor.161. The method of embodiment 160, wherein said CDK4/6 inhibitor ispalbociclib, abemaciclib, or ribociclib.162. The method of any one of embodiments 142 to 153, further comprisingadministering to said patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor.163. The method of embodiment 162, wherein said mTOR inhibitor isEverolimus.164. The method of any one of embodiments 142 to 153, further comprisingadministering to said patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor.165. The method of any one of embodiments 142 to 153, further comprisingadministering to said patient an effective amount of human epidermalgrowth factor receptor 2 (HER2) inhibitor.166. The method of embodiment 165, wherein said HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®).167. The method of any one of embodiments 142 to 153, further comprisingadministering to said patient an effective amount of a histonedeacetylase (HDAC) inhibitor.168. The method of embodiment 167, wherein said HDAC inhibitor isvorinostat (Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®),panobinostat (Farydak®), belinostat (Beleodaq®, PXD101), valproic acid(Depakote®, Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat(PCI-24781), entinostat (MS-275), pracinostat (SB939), resminostat(4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin,CUDC-101, AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745,rocilinostat (ACY-1215), or sulforaphane.169. The method of any one of embodiments 142 to 153, further comprisingadministering to said patient an effective amount of checkpointinhibitor.170. The method of embodiment 169, wherein said checkpoint inhibitor isan antibody specific for programmed cell death protein 1 (PD-1),programmed death-ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4).171. The method of embodiment 170, wherein said PD-1 antibody ispembrolizumab (Keytruda®) or nivolumab (Opdivo®).172. The method of embodiment 170, wherein said CTLA-4 antibody isipilimumab (Yervoy®).173. The method of any one of embodiments 142 to 153, further comprisingadministering to said patient an effective amount of cancer vaccine.174. The method of any one of embodiments 142 to 173, wherein thepatient is premenopausal.175. The method of any one of embodiments 142 to 173, wherein thepatient is perimenopausal.176. The method of any one of embodiments 142 to 173, wherein thepatient is postmenopausal.177. A method of treating a female patient suffering from breast cancerwho is at risk of acquiring a gain of function missense mutation withinthe ligand binding domain (LBD) of the Estrogen Receptor 1 (ESR1) gene,comprising administering to the female patient an effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof.178. A method of treating a female patient suffering from breast cancerwho is at risk of acquiring resistance to endocrine therapy, optionallywherein the endocrine therapy is (i) selective ER modulator (SERM)therapy, (ii) selective ER degrader (SERD) therapy, (iii) aromataseinhibitor (AI) therapy, or (iv) any combination of (i), (ii) and/or(iii), comprising administering to the female patient an effectiveamount of lasofoxifene, a pharmaceutically acceptable salt thereof, or aprodrug thereof.179. The method of embodiment 177 or embodiment 178, wherein the patienthas primary breast cancer.180. The method of embodiment 179, wherein the primary breast cancer islocally advanced.181. The method of any one of embodiments 177 to 180, wherein thepatient has been treated with endocrine therapy, optionally wherein theendocrine therapy is (i) selective ER modulator (SERM) therapy, (ii)selective ER degrader (SERD) therapy, (iii) aromatase inhibitor (AI)therapy, or (iv) any combination of (i), (ii) and/or (iii).182. A method of treating a female patient suffering from estrogenreceptor positive (ER+) primary breast cancer, comprising administeringto a female patient an effective amount of lasofoxifene, apharmaceutically acceptable salt thereof, or a prodrug thereof.183. The method of embodiment 182, wherein the patient is at risk ofacquiring resistance to endocrine therapy, optionally wherein theendocrine therapy is (i) selective ER modulator (SERM) therapy, (ii)selective ER degrader (SERD) therapy, (iii) aromatase inhibitor (AI)therapy, or (iv) any combination of (i), (ii) and/or (iii).184. The method of embodiment 182 or embodiment 183, wherein the primarybreast cancer is locally advanced.185. The method of any one of embodiments 182 to 184, wherein thepatient has been treated with endocrine therapy, optionally wherein theendocrine therapy is (i) selective ER modulator (SERM) therapy, (ii)selective ER degrader (SERD) therapy, (iii) aromatase inhibitor (AI)therapy, or (iv) any combination of (i), (ii) and/or (iii).186. A method of treating a female patient suffering from estrogenreceptor positive (ER+) locally advanced or metastatic breast cancer,comprising administering to a female patient an effective amount oflasofoxifene, a pharmaceutically acceptable salt thereof, or a prodrugthereof.187. The method of embodiment 186, wherein the patient has previouslybeen treated with one or more lines of endocrine therapy.188. The method of embodiment 186, wherein the patient has previouslybeen treated with a plurality of lines of endocrine therapy.189. The method of any one of embodiments 186 to 188, wherein thepatient has disease progression after endocrine therapy.190. The method of any one of embodiments 186 to 188, wherein theendocrine therapy that the patient has previously been treated with is aselective ER modulator (SERM).191. The method of embodiment 190, wherein the SERM is tamoxifen,raloxifene, bazedoxifene, toremifene, or ospemifene.192. The method any one of embodiments 186 to 188, wherein the endocrinetherapy that the patient has previously been treated with is a selectiveER degrader (SERD).193. The method of embodiment 192, wherein the SERD is fulvestrant,RAD1901, ARN-810 (GDC-0810), or AZD9496.194. The method of any one of embodiments 186 to 188, wherein theendocrine therapy that the patient has previously been treated with isan aromatase inhibitor.195. The method of embodiment 194, wherein the aromatase inhibitor isexemestane (Aromasin®), letrozole (Femara®), or anastrozole (Arimidex®).196. The method of any one of embodiments 187 to 195, wherein thepatient has disease progression after endocrine therapy.197. The method of any one of embodiments 186 to 196, wherein thepatient's locally advanced or metastatic cancer is resistant toendocrine therapy other than lasofoxifene.198. The method of any one of embodiments 186 to 197, wherein thepatient has cancer cells with at least one gain of function missensemutation within the ligand binding domain (LBD) of the Estrogen Receptor1 (ESR1) gene.199. The method of embodiment 198, wherein the patient has previouslybeen determined to have at least one gain of function missense mutationwithin the ligand binding domain (LBD) of the Estrogen Receptor 1 (ESR1)gene.200. The method of embodiment 197, further comprising the earlier stepof:

determining that the patient has at least one gain of function missensemutation within the ligand binding domain (LBD) of the Estrogen Receptor1 (ESR1) gene.

201. The method of any one of embodiments 198 to 198, wherein the atleast one of gain of function missense mutation is in any one of aminoacids D538, Y537, L536, P535, V534, S463, V392, and E380.202. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid D538.203. The method of embodiment 202, wherein the mutation is D538G.204. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid Y537.205. The method of embodiment 204, wherein the mutation is Y537S, Y537N,Y537C, or Y537Q.206. The method of embodiment 205, wherein the mutation is Y537C.207. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid L536.208. The method of embodiment 207, wherein the mutation is L536R orL536Q.209. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid P535.210. The method of embodiment 209, wherein the mutation is P535H.211. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid V534.212. The method of embodiment 211, wherein the mutation is V534E.213. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid S463.214. The method of embodiment 213, wherein the mutation is S463P.215. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid V392.216. The method of embodiment 215, wherein the mutation is V392I.217. The method of embodiment 201, wherein the at least one gain offunction missense mutation is in the amino acid E380.218. The method of embodiment 217, wherein the mutation is E380Q.219. The method of any one of embodiments 177 to 218, whereinlasofoxifene is administered as lasofoxifene tartrate.220. The method of any one of embodiments 177 to 219, whereinlasofoxifene is administered by oral, intravenous, transdermal, vaginaltopical, or vaginal ring administration.221. The method of embodiment 220, wherein lasofoxifene is administeredby oral administration.222. The method of embodiment 221, wherein lasofoxifene is administeredat about 0.5 mg/day per os to about 10 mg/day per os.223. The method of embodiment 222, wherein lasofoxifene is administeredat about 0.5 mg/day per os to about 5 mg/day per os.224. The method of embodiment 223, wherein lasofoxifene is administeredat about 1 mg/day per os to about 5 mg/day per os.225. The method of embodiment 223, wherein lasofoxifene is administeredat 1 mg/day per OS.226. The method of embodiment 223, wherein lasofoxifene is administeredat 5 mg/day per OS.227. The method of any one of embodiments 177 to 226, whereinlasofoxifene is administered once every day, once every two days, onceevery three days, once every four days, once every five days, once everysix days, once every week, once every two weeks, once every three weeks,or once every month.228. The method of any one of embodiments 177 to 227, further comprisingtreating said patient with at least one additional endocrine therapy.229. The method of embodiment 228, wherein said patient is treated withthe additional endocrine therapy at original doses.230. The method of embodiment 228, wherein said patient is treated withthe additional endocrine therapy at doses higher than original doses.231. The method of any one of embodiments 228 to 230, wherein theadditional endocrine therapy is treatment with a selective ER modulator(SERM) other than lasofoxifene.232. The method of any one of embodiments 228 to 230, wherein theadditional endocrine therapy is treatment with a selective ER degrader(SERD).233. The method of any one of embodiments 228 to 230, wherein theadditional endocrine therapy is treatment with an aromatase inhibitor.234. The method of any one of embodiments 177 to 227, further comprisingadministering to said patient an effective amount of cyclin-dependentkinase 4/6 (CDK4/6) inhibitor.235. The method of embodiment 234, wherein said CDK4/6 inhibitor ispalbociclib, abemaciclib, or ribociclib.236. The method of any one of embodiments 177 to 227, further comprisingadministering to said patient an effective amount of mammalian target ofrapamycin (mTOR) inhibitor.237. The method of embodiment 236, wherein said mTOR inhibitor isEverolimus.238. The method of any one of embodiments 177 to 227, further comprisingadministering to said patient an effective amount of phosphoinositide3-kinase (PI3K) inhibitor or heat shock protein 90 (HSP90) inhibitor.239. The method of any one of embodiments 177 to 227, further comprisingadministering to said patient an effective amount of human epidermalgrowth factor receptor 2 (HER2) inhibitor.240. The method of embodiment 239, wherein said HER2 inhibitor istrastuzumab (Herceptin®) or ado-trastuzumab emtansine (Kadcyla®).241. The method of any one of embodiments 177 to 227, further comprisingadministering to said patient an effective amount of a histonedeacetylase (HDAC) inhibitor.242. The method of embodiment 241, wherein said HDAC inhibitor isvorinostat (Zolinza®), romidepsin (Istodax®), chidamide (Epidaza®),panobinostat (Farydak®), belinostat (Beleodaq®, PXD101), valproic acid(Depakote®, Depakene®, Stavzor®), mocetinostat (MGCD0103), abexinostat(PCI-24781), entinostat (MS-275), pracinostat (SB939), resminostat(4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), kevetrin,CUDC-101, AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745,rocilinostat (ACY-1215), or sulforaphane.243. The method of any one of embodiments 177 to 227, further comprisingadministering to said patient an effective amount of a checkpointinhibitor.244. The method of embodiment 243, wherein said checkpoint inhibitor isan antibody specific for programmed cell death protein 1 (PD-1),programmed death-ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4).245. The method of embodiment 244, wherein said PD-1 antibody ispembrolizumab (Keytruda®) or nivolumab (Opdivo®).246. The method of embodiment 244, wherein said CTLA-4 antibody isipilimumab (Yervoy®).247. The method of any one of embodiments 177 to 227, further comprisingadministering to said patient an effective amount of cancer vaccine.248. The method of any one of embodiments 177 to 247, wherein thepatient is premenopausal.249. The method of embodiment 248, wherein the patient has locallyadvanced or metastatic ER+/HER2− breast cancer.250. The method of embodiment 249, wherein the patient has progressed onher first hormonal treatment while on a non-steroid aromatase inhibitor(AI), fulvestrant, AI in combination with a CDK4/6 inhibitor, orfulvestrant in combination with a CDK4/6 inhibitor.251. The method of any one of embodiments 177 to 247, wherein thepatient is perimenopausal.252. The method of embodiment 251, wherein the patient has locallyadvanced or metastatic ER+/HER2− breast cancer.253. The method of embodiment 252, wherein the patient has progressed onher first hormonal treatment while on a non-steroid aromatase inhibitor(AI), fulvestrant, AI in combination with a CDK4/6 inhibitor, orfulvestrant in combination with a CDK4/6 inhibitor.254. The method of any one of embodiments 177 to 247, wherein thepatient is postmenopausal.255. The method of embodiment 254, wherein the patient has locallyadvanced or metastatic ER+/HER2− breast cancer.256. The method of embodiment 255, wherein the patient has progressed onher first hormonal treatment while on a non-steroid aromatase inhibitor(AI), fulvestrant, AI in combination with a CDK4/6 inhibitor, orfulvestrant in combination with a CDK4/6 inhibitor.

6.6. EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of molecular biology, cell biology,biochemistry, genetics, cancer biology, and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.

6.6.1. Example 1: Efficacy of Lasofoxifene on ESR1 LBD Mutations6.6.1.1. Methods 6.6.1.1.1. Site-Directed Mutagenesis

ExSite mutagenesis was performed using the corresponding primers assummarized in Table 1 below on a pENTR2B ERα WT construct using Pfuultra taq polymerase. The primers were PNK phosphorylated. Following PCRamplification, the products were digested with DpnI at 37° C. for 1 hr,followed by overnight ligation at 16° C. Ligated products weretransformed into DH5α bacterial cells and grown on kanamycin resistantplates. The pENTR clones were verified by sequencing and then swappedinto the pcDNA-DEST vector using the Gateway system (Invitrogen) forexpression analysis.

TABLE 1 Primers for Mutagenesis ER Y537N For AATGACCTGCTGCTGGAGATGSEQ ID NO: 1 ER Y537N Rev GAGGGGCACCACGTTCTTGCA SEQ ID NO: 2ER Y537S For GACCTGCTGCTGGAGATGCTG SEQ ID NO: 3 ER Y537S RevGCTGAGGGGCACCACGTTCTT SEQ ID NO: 4 ER Y537C For TGTGACCTGCTGCTGGAGATGSEQ ID NO: 5 ER Y537C Rev GCTGAGGGGCACCACGTTCTT SEQ ID NO: 6ER D538G For GGTCTGCTGCTGGAGATGCTG SEQ ID NO: 7 ER D538G RevATAGAGGGGCACCACGTTCTT SEQ ID NO: 8

6.6.1.1.2. Cell Culture

Caov2 ovarian carcinoma cells were grown in RPMI-1640 media (Gibco)supplemented with 8% Fetal Bovine Serum (FBS), Sodium Pyruvate (NaPyr)and non-essential amino acids (NEAA) and passaged every 2-3 days. SKBR3breast adenocarcinoma cells were grown in DMEM media (Gibco)supplemented with 8% Fetal Bovine Serum (FBS), Sodium Pyruvate (NaPyr)and non-essential amino acids (NEAA) and passaged every 2-3 days. Cellswere switched into a phenol-red free RPMI-1640 media supplemented with8% charcoal stripped fetal bovine serum (CFS), NaPyr, and NEAA one daybefore plating for experiment. Cells were then plated in 96-well platesfor experiment in the phenol red-free media an additional day beforetransfection.

6.6.1.1.3. Reporter Gene Assay

Caov2 cells were co-transfected with the 7X-TK-ERE-TATA luciferasereporter gene (Nagel et al., Endocrinology 142(11): 4721-4728 (2001))and expression constructs for either wild-type or mutant receptors usingFugene transfection reagent (Promega). SKBR3 cells were co-transfectedwith 3X-TK-ERE-TATA luciferase reporter gene in the same conditions.pCMV-β-gal was used as a control for transfection efficiency and pcDNAwas added for a final DNA concentration of 75 ng per triplicate group.Cells were treated with indicated ligand five hours post transfection.Following 24 hours of treatment, cells were lysed and the luciferase andβ-gal assays were performed as described previously (Norris et al., JBiol Chem 270(39): 22777-22782 (1995)) and the plates were read on theFusion α-FP HT plate reader (PerkinElmer Life Sciences).

6.6.1.2. Results

ERα expression constructs were engineered to express one of fourdifferent ESR1 LBD mutations, Y537S, Y537N, Y537C, and D538G, which arefound in metastatic breast cancer patients. See Jeselsohn et al., NatureReviews Clinical Oncology 12(10): 573-583 (2015); Jeselsohn et al.,Clinical Cancer Research 20(7): 1757-1767 (2014); Robinson et al.,Nature Genetics 45(12): 1446-1451(2013); Thomas and Gustafsson, Trendsin Endocrinology and Metabolism 26(9): 467-476 (2015); and Toy et al.,Nature Genetics 45(12): 1439-1445 (2013). The activity of these mutantswas evaluated in a reconstituted estrogen response element(ERE)-luciferase reporter assay in Caov2 ovarian carcinoma cells andSKBR3 breast adenocarcinoma cells. Data normalization is done in respectto the “0” data point (no ligand) of the wild-type receptor. Aspreviously reported (Jeselsohn et al., 2014; Robinson et al., 2013; Toyet al., 2013), all of the mutants studied exhibited substantialconstitutive activity when compared to the activity of wild-type (WT)ERα in the absence of its ligand: 17-β estradiol (E2). While the WT ERαresponds to E2 in a dose-response matter, the transcriptional activityof the mutants is not responsive to E2 activation (FIG. 1A and FIG. 2A).

The ability of lasofoxifene to inhibit the transcriptional activity ofthe ERα mutants was next evaluated under the same conditions. Allinhibition curves were done in the presence of 10⁻⁹ (1 nM) 17-βestradiol. Data normalization was done in respect to the “0” data point(no lasofoxifene) for each individual receptor. The plots include datafrom five independent experiments and each value is an average oftriplicates from each experiment. Notably, lasofoxifene effectivelyinhibited the transcriptional activity of all tested ERα LBD mutants ina dose-response manner (FIG. 1B and FIG. 2B).

The transcriptional IC90 value of lasofoxifene was also evaluated underthe same conditions in Caov2 ovarian carcinoma cells and SKBR3 breastadenocarcinoma cells. See Maximov et al., Current Clinical Pharmacology8(2): 135-155 (2013). The transcriptional IC90 value of lasofoxifeneevaluated was compared to the Cmax of these compounds in blood at dosesused in prior clinical trials and approved in Europe. See AssessmentReport for Fablyn, 2009 (EMA). The calculation included Cmax oflasofoxifene at theoretical doses of 0.5 mg and 1 mg. The additionaldose of lasofoxifene (1 mg) was included to evaluate the potentialclinical efficacy of lasofoxifene at a higher concentration. See Gardneret al., J Clin Pharmacol 46(1): 52-58 (2006). The results from Caov2ovarian carcinoma cells and SKBR3 breast adenocarcinoma cells aresummarized in Table 2.

TABLE 2 Comparison of IC90 Values to Reported Cmax Values Caov2 SKBR3Reported Converted (M) Caov2 Ratio SKBR3 Ratio Compound Cmax Cmax IC90Cmax/IC90 IC90 Cmax/IC90 WT Lasofoxifene (0.5 mg)  3.6 ng/mL 9.00E−096.68E−12 1346.8 3.30E−09 2.73 Lasofoxifene (1 mg) 6.43 ng/mL 1.55E−086.68E−12 2320.4 3.30E−09 4.69 Y537N Lasofoxifene (0.5 mg)  3.6 ng/mL9.00E−09 7.45E−10 12.08 1.30E−08 0.69 Lasofoxifene (1 mg) 6.43 ng/mL1.55E−08 7.45E−10 20.8 1.30E−08 1.19 Y537S Lasofoxifene (0.5 mg)  3.6ng/mL 9.00E−09 1.22E−08 0.74 8.00E−09 1.13 Lasofoxifene (1 mg) 6.43ng/mL 1.55E−08 1.22E−08 1.27 8.00E−09 1.94 Y537C Lasofoxifene (0.5 mg) 3.6 ng/mL 9.00E−09 2.04E−10 44.07 5.90E−09 1.53 Lasofoxifene (1 mg)6.43 ng/mL 1.55E−08 2.04E−10 75.98 5.90E−09 2.63 D538G Lasofoxifene (0.5mg)  3.6 ng/mL 9.00E−09 1.88E−09 4.80 7.10E−09 1.27 Lasofoxifene (1 mg)6.43 ng/mL 1.55E−08 1.88E−09 8.24 7.10E−09 2.18

As expected, the WT receptor was the most responsive to anti-estrogentreatment, with each of the mutants exhibiting reduced response to theinhibitory actions of lasofoxifene. Importantly, the pharmacology ofeach of the mutants was different, which highlights the need to matchpatients with the most appropriate drug. The data suggest thatlasofoxifene at a dose of 1 mg is most effective for patients whosetumors express the mutations in both ovarian and breast cancer settings.

6.6.2. Example 2: Efficacy of Lasofoxifene on ESR1 LBD Mutations Y537Sand D538G in Stable Transfectants

MCF7 estrogen receptor alpha positive (ER⁺) breast cancer cells wereengineered to stably express doxycycline (DOX)-inducible hemagglutinin(HA)-tagged full length ER with ligand binding domain mutations Y537Sand D538G. The introduction and expression of the mutants were confirmedby Sanger sequencing, RNA-sequencing, and western blot.

The dose response studies were performed in full medium conditions.Cells were treated with DOX for the induction of HA-tagged mutated ER orwith vehicle as control, and plated in triplicate. Subsequently, on day5, cell counting was performed using the Celigo instrument with Hoechstdye staining to detect nucleated live cells and propidium iodide toquantify dead cells. Treatments included vehicle and increasing doses oflasofoxifene starting from 10⁻¹²M with 10 fold increments up to 10⁻⁶M.The efficacy of the treatment is inversely proportional to the cellcount.

The anti-estrogenic activity of lasofoxifene in a breast cancer model ofER mutations Y537S and D538G identified in Example 1 was confirmed bythe ability of lasofoxifene to overcome resistance with increasing dosetitration and kill the stably transfected cells (FIG. 3A and FIG. 3B).

IC50 values were calculated using PRISM. The results are summarized inTable 3.

TABLE 3 Comparison of IC50 Values in the Absence and the Presence of DOXNo DOX DOX Treatment Allele (wt only) (ESR mutation) Fold ChangeLasofoxifene Y537S 3.6E−10 4.1E−9 11.4 Lasofoxifene D538G  1E−10  1E−910

The results confirmed that lasofoxifene treatment is effective on theY537S and D538G mutations, although the Y537S and D538G mutationsrequire higher concentrations to overcome resistance.

7. EQUIVALENTS AND INCORPORATION BY REFERENCE

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. A method for treating estrogen receptor positive and human epidermalgrowth factor receptor 2 negative (ER⁺/HER2⁻) advanced or metastaticbreast cancer in a pre-menopausal or post-menopausal woman patient,wherein the cancer has at least one mutation in the Estrogen Receptor 1(ESR1) gene and had progressed following treatment with an aromataseinhibitor (AI) in combination with the cyclin D 4/6 kinase (CDK4/6)inhibitor palbociclib or the CDK4/6 inhibitor ribociclib, the methodcomprising: orally administering to the patient 5 mg lasofoxifene perday and abemaciclib.
 2. The method of claim 1, wherein lasofoxifene isadministered as lasofoxifene tartrate.
 3. The method of claim 1, whereinthe at least one ESR1 mutation is in any one of amino acids D538, Y537,L536, P535, V534, S463, V392, and E380 of the encoded ERα protein. 4.The method of claim 3, wherein the at least one ESR1 mutation is D538G,Y537S, Y537N, Y537C, Y537Q, L536R, L536Q, P535H, V534E, S463P, V392I, orE380Q.
 5. The method of claim 4, wherein the at least one ESR1 mutationis D538G, Y537S, Y537N, or Y537C.
 6. The method of claim 1, wherein theESR1 mutation is detected from cell-free circulating tumor DNA (ctDNA).7. The method of claim 1, wherein the aromatase inhibitor treatment onwhich the cancer had progressed was treatment with exemestane,letrozole, or anastrozole.
 8. The method of claim 1, wherein the canceris locally advanced.
 9. The method of claim 1, wherein the cancer ismetastatic.